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Latest Articles of Bio-Synthesis Inc.

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    Neurodegeneration and Neurodegenerative Diseases

    What do we fear the most when we age? Most of us will most like fear the degeneration of our brain power the most. I admit that symptoms like forgetting names or places that I know I knew annoys me a lot. Therefore neurodegenerative disorders are some of the most feared illnesses in our society.

     

    Alzheimer’s disease affects up to 10% of people over 65 years of age. This disease causes the progressive loss of memory and other mental faculties, leaving the individual confused and incompetent to care for him- or herself. Huntington’s disease is another relatively common neurodegenerative disorder. One in ten thousand individuals are affected. People inflicted with this disease make involuntary movements and become serverely emotionally disturbed as well as cognitively impaired. Prion diseases, including Creutzfeldt-Jakob disease, lead to mental and physical decline followed by death.

     

    Many scientists are working and have been working to find a treatment or cure, still, no successful treatments are yet available for any of this diseases but some progress has been made during recent years. Scientific research in this field aims to understand the disease mechanism in order to develop successful treatments and to prevent the onset of symptoms in patients. Particular genetic traits appear to be linked to many of these diseases. For example, the chromosome and gene linked to Huntington’s disease, Freiderich’s ataxia, the prion genes linked to spongiform encephalopathies, as well as the triplet repeat mutations responsible for myotonic dystrophy have all been identified. Model systems in which to test potential therapies and prevention strategies have been developed and are employed to find a cure for this cruel and distressing diseases.

     

    Familiar neurodegenerative disorders are Alzheimer’s, Parkinson’s, Huntington’s and Wilson’s disorders. Table 1 shows a list of autosomal neurodegenerative diseases including what is known about chromosomal linkage, genes, mutations and pathology.

     

    Table 1. Autosomal dominant primary neurodegenerative diseases. Ch, chromosome; PrP, prion protein; T, tangles; LB, Lewy bodies; 1, is present or exists; AD, Alzheimer’s disease; PD, Parkinson’s disease; HD, Huntington’s disease; SOD, superoxide dismutase.


    Disease Linkage Gene Mutations Pathology Transgenic (comment)
    Prion disease Ch20 Prion Mainly missense PrP plaques, sometimes T or LB; classically associated with spongiform changes   + (no T or LB)
    Alzheimer’s disease (AD) Ch21 APP Missense around Ab, increase Ab42 Amyloid plaques and T, may see LB + (no T or LB)
    Alzheimer’s disease Ch14 PS1 Mainly missense, increase Ab42 Amyloid plaques and T + (no plaques T or LB)
    Alzheimer’s disease Ch1 PS2 Missense, increase Ab42 Amyloid plaques and T + (no plaques T or LB)
    Parkinson’s disease (PD) Ch4q a-synuclein Missense LB Not reported
    Parkinson’s disease Ch2 Not identified Not known LB (and T?) Not reported
    Parkinson’s disease Ch4p Not identified Not known LB Not reported
    Frontotemporal dementia (FTD) Ch17 Tau Missense and splice T, sometimes with “unusual Periodicity”   Not reported
      Ch3 Not identfied Not known Not reported Not reported
    Amyotrophic lateral sclerosis (ALS) 14 types Ch21 SOD Mainly missense   110 different mutations Lewy-like bodies + (motor neuron disease, inclusions, cell loss)
    Familial amyotrophic lateral sclerosis (FALS) Hexa nucleotide repeat abnormality C9ORF72      
    Spinal and bulbar muscular atrophy (SBMA) X AR Polyglutamine Nuclear inclusions + (no phenotype)
    Huntington's disease HD Ch4 Huntingtin Polyglutamine Nuclear inclusions +  (inclusions, movement disorder, cell loss)
    Dentatorubral-pallidoluysian atrophy (DRPLA) Ch12 Atrophin 1 Polyglutamine Nuclear inclusions Not reported
    Spinocerebellar ataxia (SCA1) Ch6 Ataxin 1 Polyglutamine Nuclear inclusions + (ataxic, inclusions, cell loss)
    SCA2 Ch12 Ataxin 2 Polyglutamine Not reported Not reported
    SCA3/MJD Ch14 Ataxin 3 Polyglutamine Nuclear inclusions + (ataxic, cerebellar atrophy)
    SCA4 Ch16 Not identified Not known Not reported Not reported
    SCA5 Ch11 Not identified Not known Not reported Not reported
    SCA6 Ch19 CACNL1A4 Polyglutamine Not reported Not reported
    SCA7 Ch3 SCA7 Polyglutamine Nuclear inclusions Not reported
               

    *SBMA is technically not autosomal dominant but it is probably dominant in its cellular mode of action.
     

    Alzheimer's disease (AD) is the most common form of dementia among older people which is a brain disorder that seriously affects a person's ability to carry out daily activities. AD begins slowly, it first involves the parts of the brain that control thought, memory and language. Over time, symptoms get worse. People may not recognize family members or have trouble speaking, reading or writing. They may forget how to brush their teeth or comb their hair. Later on, they may become anxious or aggressive, or wander away from home. Eventually, they need total care. The disease usually begins after age 60 and the risk of getting it goes up with age. The risk is also higher if a family member has had the disease. No treatment can yet stop the disease.

     

    Amyotrophic lateral sclerosis (ALS) is a disease that attacks nerve cells called neurons in the brain and spinal cord. The job of neurons is to transmit messages from the brain and spinal cord to voluntary muscles - the ones that control arms and legs. At first, this causes mild muscle problems, some people notice trouble when walking or running, writing and may notice speech problems. Eventually they lose strength and cannot move. When the muscles in the chest fail they cannot breathe. A ventilator may help, but most people with ALS die from respiratory failure. The disease usually strikes between age 40 and 60. More men than women get it. No one knows what causes ALS. It can run in families, but usually it strikes at random. There is no cure. Medicines can relieve symptoms and, sometimes, prolong survival.

     

    Friedreich's ataxia is an inherited disease that damages the nervous system. It affects the spinal cord and the nerves that control muscle movement in arms and legs. Symptoms usually begin between the ages of 5 and 15. The main symptom is ataxia, which is trouble coordinating movements. Specific symptoms include difficulty to walk, muscle weakness, speech problems, involuntary eye movements, scoliosis and heart palpitations. People with Friedreich's ataxia usually need a wheelchair 15 to 20 years after symptoms first appear. In severe cases, people become incapacitated. There is no cure.

     

    Huntington's disease (HD) is an inherited disease that causes certain nerve cells in the brain to waste away. People are born with the defective gene, but symptoms usually don't appear until middle age. Early symptoms of HD may include uncontrolled movements, clumsiness or balance problems. Later, HD can take away the ability to walk, talk or swallow. Some people stop recognizing family members. Others are aware of their environment and are able to express emotions. If one of the parents has Huntington's disease, there is a 50-50 chance of getting it. A blood test can tell if the HD gene is present and if there is a risk to develop the disease. There is no cure. Medicines can help manage some of the symptoms, but cannot slow down or stop the disease.

     

    Lewy body disease is one of the most common causes of dementia in the elderly that causes a loss of mental functions severe enough to affect normal activities and relationships. Lewy body disease happens when abnormal structures, called Lewy bodies, build up in areas of the brain. The disease may cause a wide range of symptoms, including changes in alertness and attention, hallucinations, problems with movement and posture, muscle stiffness, confusion and finally loss of memory. Lewy body disease can be hard to diagnose since it can be confused with Parkinson’s and Alzheimer’s disease. The disease usually begins between the ages of 50 and 85. The disease gets worse over time. There is no cure.

     

    Parkinson's disease affects nerve cells, or neurons, in a part of the brain that controls muscle movement. In Parkinson's, neurons that make a chemical called dopamine die or do not work properly. Dopamine normally sends signals that help coordinate movements. Symptoms of Parkinson's disease may include trembling of hands, arms, legs, jaw and face, stiffness of the arms, legs and trunks, slowness of movement, poor balance and coordination. Parkinson's usually begins around age 60 but can start earlier. It is more common in men than in women. There is no cure for Parkinson's disease.

     

    Spinal muscular atrophy (SMA) attacks nerve cells, called motor neurons, in the spinal cord. These neurons communicate with voluntary muscles. As neurons are lost muscles weaken. This can affect walking, crawling, breathing, swallowing and head and neck control. SMA runs in families. Parents usually have no symptoms, but still carry the gene. There are many types of SMA, and some of them are fatal. Life expectancy depends on the type of disease.

     

    Spinocerebellar ataxia (SCA) a progressive and degenerative genetic disease containes multiple types, each of which could be considered a disease in its own right. The following is a list of some, not all, types of Spinocerebellar ataxia. The first ataxia gene was identified in 1993 for a dominantly inherited type. It was called “Spinocerebellar ataxia type 1" (SCA1). Subsequently, as additional dominant genes were found they were called SCA2, SCA3, etc. Usually, the "type" number of "SCA" refers to the order in which the gene was found. At this time, there are at least 29 different gene mutations which have been found. Many SCAs below fall under the category of polyglutamine diseases, which are caused when a disease-associated protein (i.e. ataxin-1, ataxin-3, etc.) contains a glutamine repeat beyond a certain threshold. In most dominant polyglutamine diseases, the glutamine repeat threshold is approximately 35, except for SCA3 which is beyond 50. Polyglutamine diseases are also known as "CAG Triplet Repeat Disorders" because CAG is the codon which codes for the amino acid glutamine. Many prefer to refer to these also as polyQ diseases since "Q" is the one-letter reference for glutamine.

     

    Spinal and bulbar muscular atrophy (SBMA), also known as spinobulbar muscular atrophy, X-linked bulbo-spinal atrophy, X-linked spinal muscular atrophy type 1 (SMAX1) and Kennedy's disease (KD)— A X-linked, recessive, slow progressing, neurodegenerative disease associated with mutations of the androgen receptor (AR) gene resulting in the impairment of the AR that can be viewed as a variation of the disorders of the androgen insensitivity syndrome (AIS).

     

    Dentatorubral-pallidoluysian atrophy (DRPLA) is an autosomal dominant pinocerebellar degeneration caused by an expansion of a CAG repeat encoding a polyglutamine tract in the atrophin-1 protein. It is also known as Haw River Syndrome and Naito-Oyanagi disease. Although this condition was perhaps first described by Smith et al. in 1958, and several sporadic cases have been reported from Western countries, this disorder seems to be very rare except in Japan.

     

    There are at least eight neurodegenerative diseases that are caused by expanded CAG repeats encoding polyglutamine (polyQ) stretches. The expanded CAG repeats create an adverse gain-of-function mutation in the gene products. Of these diseases, DRPLA is most similar to Huntington disease.


    References
     

    John Hardy and Katrina Gwinn-Hardy Genetic Classification of Primary Neurodegenerative Disease. SCIENCE VOL 282 6 NOVEMBER 1998, 1075-1079.

    NIH: National Institute on Aging.

    NIH: National Institute of Neurological Disorders and Stroke.


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  • 11/05/12--00:00: Amino Acid Analysis
  • Amino Acid Analysis

    0. Introduction

    The analysis of free amino acids present in food samples, body fluids such as urine, serum and blood, and other sources, amino acid hydrolysates, from proteins, or primary and secondary amines is an important, standardized method routinely performed in biochemical, medical and biological labs. It has been, and still is used for the accurate quantification and characterization of proteins and peptides, as well as recombinant gene products. It is considered the method of choice to determine the purity and chemical composition of a protein or peptide.

    1. Types of amino acid analyses

    A. Hydrolysate analyses

    Composition studies: Determination of amino acid content of proteins, peptides, foods, beverages, cosmetics, and others.
    Quality control:             Verification of product composition

    B. Physiological analyses

    Nutrition studies:       Determination of free amino acid content in food supplements, and others.
    Clinical assays:         Determination of amino acid content in serum, tissue, and other body fluids.

         Amino acid analysis (AAA) is a method for breaking down a protein or peptide into its components (amino acids) and determining their identities and relative quantities of the freed amino acids. Absolute quantities of amino acids released from the protein or peptide can also be determined.

         AAA can be used in combination with protein sequence analysis to verify if the total sequence of the protein in question has been determined. It also helps to identify modified amino acids like phosphor-tyrosine, the presence of amino acids modified with carbohydrates, if amino sugars are found, and others.

         Knowledge of the number of methionines or tryptophanes present allows for the design of peptide mapping strategies employing enzymatic or chemical cleavage methods to generate a limited number of longer peptides. These peptides may then be sequenced and used for the design of primers. Knowledge of the number of Lys, Arg, Asp, and Glu residues allows one to select the most appropriate protease for further experiments.

         Even today, AAA is still the most practical method for accurately quantifying amino acid, peptide and protein concentrations. Accurate measurements are essential for calculating extinction coefficients of proteins or to determine the turnover number for an enzyme. Although instrumentation performance has improved over the years, the basic concepts of amino acid analysis have not changed since Stein and Moore (1963) developed the original method based on ion exchange resins.

    2. Sample preparation

         The preparation of samples to be used in AAA can be a quite difficult but very important experimental step for a successful amino acid analysis. Contamination with other proteins, amino acids, and other molecular weight solutes that interfere with the analysis chemistry are the most serious. To get accurate results the purity of the sample is much more critical than for sequencing or mass analysis.

    2.1. Contamination with traces of undesired proteins:

         In the case of proteins a 10% contamination of the desired protein with an undesired byproduct such as a different protein may render the data useless.

    2.2. Low molecular weight compounds

         Compounds containing primary or secondary amino groups are a very serious problem. They can be found in abundance in every laboratory and are difficult to completely eliminate. Tris and glycine are two good examples. Contamination is the major factor limiting increases in the sensitivity of analysis. The rule of thumb here is: the more steps used prior to analysis the more contamination one can pick up.

    2.3. Non-amines

    Non-amine compounds including many buffers, detergents, and inorganic salts, especially high salt concentrations may interfere with the analysis and need to be removed prior to analysis.

    2.4. Sample treatment for physiological samples

         Physiological samples need to be treated differently than proteins prior to loading on to the derivatizer unit. To avoid losses of labile amino acids present in the sample, keep sample solutions on ice and store in freezer below -20 ºC between use or better (if enough sample is at hand), prepare aliquots and store frozen in freezer prior to analysis.

    Precipitation of proteins with 5-sulfocalicylic acid (SSA):Human urine, serum and rat brain tissue extracts are treated with sulfosalicylic acid to precipitate protein: 20 µl of 35% sulfosalicylic acid is added to 200 µl of each sample.  These solutions are vortexed and allowed to sit at room temperature at least 20 minutes before proceeding.  The samples are then spun in a microfuge for 2 minutes and the supernatants are collected. Collagen samples are hydrolyzed in 6 N HCl, 110 oC for 24 hours.  The hydrolysates are then dried down and resuspended in 250 µg/ml K4EDTA.  Ant hemolymph does not need to be pretreated before analysis. Samples are loaded onto the analyzer as follows:  Urine - 10 µl of a 1:2 dilution of the supernatant, Serum - 10 µl of undiluted supernatant, Rat brain extract - 10 µl of the undiluted supernatant, Collagen hydrolysate - 1.2 µg in 15 µl,  Fire ant hemolymph - 4 µl of a 1:9 or 1:10 dilution. Our current knowledge that approximately 30,000 human genes appear to code for up to 1 million or more proteins has generated new interest in independent ‘de novo’ protein and peptide sequencing of gene products. Two methods are available for this task, the classical Edman chemistry based method, or the newer, more recent method which utilizes LC-MS/MS based sequencing. The second method is considered to be faster and more sensitive.

         When sufficient quantities are at hand, samples may be desalted by dialysis or size exclusion chromatography using deionized water or a volatile buffer like 1 N acetic acid. These two methods are not recommended to be used for quantities below 1 nanomole. Reversed phase-HPLC may be used for smaller sample quantities. But even this method can be tricky.

    3. Hydrolysis methods

         The second part of the analysis is the hydrolysis process. Many methods have been investigated to ensure optimal recoveries for the different amino acids analyzed.

    3a. Standard hydrolysis conditions are 6N HCl from 20 to 96 hours at 110 ºC in vacuo.

    Limitations and recovery improving modifications of the method are listed below:

     

       A single hydrolysis can not yield quantitative recoveries for all amino acids present in the sample.
       Losses of up to 50-100% can be experienced for some amino acids.

       Adding reductants and/or scavengers to the hydrolysis acid will improve yields of amino acids sensitive to hydrolysis conditions (ser, thr, met, tyr).

     

       Shorter hydrolysis times and/or lower hydrolysis temperatures will improve yields of amino acids sensitive to hydrolysis conditions but may obscure others.
    3b. Hydrolysis of sensitive amino acids

       Serine and threonine

     

         Side chain hydroxyl group is modified during hydrolysis (eg. esterification, dehydration). Typical losses using standard hydrolysis conditions are 15-20% for serine and 10-15% for threonine.

         A typical method for quantitation is to run multiple hydrolyses at different hydrolysis times and plot the serine and threonine recovery versus length of hydrolysis (hydrolyzed for 30, 60 and 90 min). Extrapolate the recovery to time = 0 to yield an accurate quantitation.

       Tyrosine

     

         Typical losses are 15-20% during hydrolysis (actual losses may be higher depending on the quality of acid used and sample amount). Side chain phenol group is attacked by traces of hypochlorite/chlorine free radicals present in HCl.      Addition of scavengers is necessary to protect tyrosine. Typically phenol is added to the acid (0.1 to 1% by weight). The quality of the phenol is important. Poor quality phenol will not protect tyrosine.

     

      Methionine

     

         Losses of methionine can vary depending on sample amount, quality of HCl, amount of oxygen present in the hydrolysis vessel, length of time the hydrolyzed sample is exposed to air on the sample slide etc. Side chain thioether is oxidized forming methionine sulfone and sulfoxide.

     

         Addition of a reductant/scavenger improves methionine yields. Choice of reductants needs to be done carefully. Some reductants (thioglycolic acid, DTT, 2-mercapto-ethanol) react with PITC through their free sulfhydryl group and generate peaks that can interfere with the PTC-amino acid analysis. Borane-DIEA should not be used with the hydrolyzer.

     

      Cysteine/Cystine

     

         Losses can be 50% or greater. The free sulfhydryl and disulfide groups are sensitive to a variety of side reactions during hydrolysis.

     

         It is necessary to reduce the disulfide bonds and alkylate or oxidize the free sulfhydryl groups generated. Derivatization to form pyridylethyl or carboxy-methyl cysteine or oxidation to cysteic acid are typical techniques to quantitate this amino acid. One recommended technique is derivatization using 4-vinyl pyridine to form pyridylethyl cysteine.

     

      Tryptophan

     

         Mostly destroyed during hydrolysis by attack on the carbon double bond in the indole ring.

     

         It is not possible to get quantitative recovery of tryptophan from samples using acid hydrolysis. In manual hydrolysis the addition of thioglycolic acid to the acid (5-15% by volume) has given 70% recovery of trp at 500 pmol. Thioglycolic acid does generate a large interfering artifact peak in the PTC-chemistry. A method using dodecanthiol as a scavenger has proven to be useful.

     

      Asparagine and Glutamine

     

         Quantitatively recovered as aspartic and glutamic acid respectively.

    Many attempts have been made over the years to improve recoveries of hydrolysis sensitive amino acids as well as to generate overall quantitative recoveries for all studied amino acids. A collection of hydrolysis conditions studied is listed in table 1.

    Table 1:   Protein/Peptide Hydrolysis Methods

     

     

    Method & comments Conditions Refs
    1.  Standard method 6N HCl, 110 oC  24-96h in vacuo  
    2. improved Trp, Cys, Thr, Ser, Tyr recovery 6N HCl +/- Phenol, 110oC  20-24h in vacuo, 150oC  1-4h in vacuo Moore & Stein (1963)
    3. improved Met, Cys, Tyr recovery 6N HCL-Na2SO3 , 110oC 24h in vacuo Swadesh et al. (1984)
    4. improved Trp, Cys, Thr, Ser, Tyr recovery HCl/Propionic acid (1:1); 150 - 160oC 15 min, 130oC 2h in vacuo Westall & Hesser (1974)
    5. improved Trp, Cys, Thr, Ser, Tyr recovery HCl/TFA (2:1), 166ºC 25 min in vacuo Tsugita & Scheffler (1982)
    6. improved Trp, Cys, Thr, Ser, Tyr recovery HCl/TFA (2:1), 5% (v/v) thioglycolic acid, 166ºC 25 min in vacuo Yokote, et al., (1986)
    7. improved Trp, recovery 6 N HCl, 0.5-6% (v/v) thioglycolic acid, 110oC 24-64h in vacuo Matsubara & Sasaki (1969)
    8. improved Trp, recovery 3N p-Toluenesulphonic acid  110oC 24-72 h in vacuo

     

    Lui & Chang (1971)
    9. improved Trp, recovery 3N Mercaptoethanesulfonic acid, 110oC 24-72 h in vacuo Penke et al., (1980)
    10. Phosphoamino acids

     



    O-phospho-Ser/Thr

    O-phospho-Tyr

    O-phospho-Ser, Thr & Tyr


    1-3 N NaOH, 37-50oC 3-18 h; or 6N HCl 2 h and 4 h, respectively at 110oC 1 h, 110oC,
    6N HCl or 5N KOH 155oC for 30 min

    6 N HCl, 110oC 1-4 h


    Kemp (1980)

    Martensen (1982)

    Capony & Demaille (1983)

    4. Derivatization methods for the amino acid analysis of proteins and peptides

          Many pre-column derivatization chemistries have been investigated over the years. All of them suffer from major disadvantages such as incompatibility with aqueous samples or dissolved salts, or interference from reagent peaks in the analysis chromatogram. A list of derivatization methods commonly used is shown in the next table:

    Table 2:     Derivatization Chemistries for Amino Acids and Amine Analysis

    Method Compound Detection mode
    1.   PITC Phenylisothiocyanate Pre-column derivatization,
    UV-detection
    2.   OPA Orthophthaldehyde Pre-column derivatization,
    fluorescent and chemilumin-escent detection
    3.    FMOC-Cl fluorenyl methyl chloroformate Pre-column derivatization,
    fluorescent and chemiluminescent detection
    4.    OPA/FMOC-Cl combined OPA/Fmoc-Cl Pre-column derivatization,
    fluorescent and chemiluminescent detection
    5.   DABS 4-dimethylaminoazo-benzene-4-sulfonyl (dabsyl) chloride Pre-column derivatization,
    detection: visible light
    6.   AQC, AccQ•Tag 6-aminoquinolyl-N-hydroxy-succinimidyl carbamate Pre-column derivatization, fluorescent detection
    7.   Ninhydrin Ruhemans purple Post-column derivatization,
    detection: visible light (440 and 570 nm).


          A "pre-column" derivatization method used by several companies which produce and marked amino acid analyzer typically consists of several steps as listed below:
    1. Derivatization of amino acids
    2. Separation of derivatized amino acids by reversed phase chromatography
    3. Detection in UV or using fluorescence for increased sensitivity (some chemistries).

          A "post-column" derivatization method mainly used for on-line ninhydrin derivatization in an automated amino acid analyzer consists of several steps as listed below:
    1. Separation of amino acids by ion exchange chromatography
    2. Derivatization of amino acids with ninhydrin at an elevated temperature
    3. Detection of derivatives via absorption in the visible range (440 and 570 nm).

    The ninhydrin based system has been the most widely used system.

    4.1. Derivatization of free amino acids using phenylisothiocyanate (PITC)


    Figure 1: Derivatization reaction of amines and free amino acids using PITC.
    PITC reacts with the free amino groups in amines and amino acids to form the phenylthiourea adduct of these compounds making them suitable for UV-detection.This reaction is used in the automated hydrolyzer/derivatizer set-up with on-line HPLC separation of the resulting PTC-amino acids and UV detection. Detection is done at 268 to 270 nm.
    4.2 Derivatization of free amino acids using ortho-phthalaldehyde (OPA)


    Figure 2: Derivatization reaction of amines and free amino acids using ortho-phthalaldehyde (OPA).
    OPA reacts with the free amino groups in aminesand amino acids in the presents of a reducing reagent like b-mercaptoethanol to form their isoindole-derivatieves making them suitable for UV-and fluorescence detection.This reaction is usually used in a pre-column derivatization step with an automated derivatizer set-up with on-line HPLC separation. UV detection is done at 338 nm. Fluorescence detection is done using excitation settings at 340 nm and emmission settings at 450 nm.
    4.3. Derivatization of free amino acids using (FMOC-Cl)


    Figure 3: Derivatization reaction of amines and free amino acids using 9-fluorenylmethyl-chloroformat (Fmoc).
    Fmoc reacts with the free amino groups in amines and amino acids to form Fmoc-derivatieves making them suitable for UV-and fluorescence detection.This reaction is usually used in a pre-column derivatization step with an automated derivatizer set-up with on-line HPLC separation. UV detection is done at 262 nm. Fluorescence detection is done using excitation settings at 266 nm and emmission settings at 305 nm.

    4.4. Derivatization using Waters AccQ•TagTM amino acid analysis system


    Figure 4A: AQC (6-aminoquinolyl-N-hydroxy-succinimidyl carbamate. Chemical structure (left) and energy minimized molecular model (right). Calculations were done using the MNDO module from CACHE Scientific.


    Figure 4B: Derivatization Chemistry.  Both 1º and 2° amino acids and amines react rapidly with AQC to produce highly stable, fluorescent derivatives. The excess reagent reacts with water to form a free amine having significantly different fluorescence spectral properties.



    References

    Barkholt, V. and Jensen, A., Anal. Biochem., (1989) 177, 318-322.

    Betner, I./ Foldi, P. New Automated Amino Acid Analysis by HPLC Precolumns Derivatization with Fluorenylmethyloxycarbonylchloride. Chromatographia Vol. 22, No. 7-12, Dec. 1986 -OPA/FMOC

    Capony and Demaille, Anal. Biochem. 152, 206-212 (1983).

    Carlson, R./ Srinivasachar, K./ Givens, R./ Matuszewski, B. New Derivatizing Agents fore Amino Acids and Peptides. 1. Facile Synthesis of N-Substituted 1-Cyanobenz[f]isoindoles and their Spectroscopic Properties. American Chemical Society (1986) 51, pg. 3978. -NDA

    Chan, King/ Janini, George/ Muschik, Gary/ Issaq, Haleem.  Laser-induced fluorescence detectin of 9-fluorenylmethyl chloroformate derivatized amino acids in capillary electrophoresis.  Journal of Chromatography A, 653 (1993) 93-97 -OPA/FMOC-Cl/NDA

    Cohen, Phillip/ Hubbard, Michael.  On target with a new mechanism for the regulation of protein phosphorylation.  TIBS 18, May 1993 pgs. 172-177.

    Cohen, S. and Michaud, D., Anal. Biochem., (1993)

    Cohen, s., and Strydom, D., Anal. Biochem., (1988) 174, 1-16.

    Cohen, Steven/ Tarvin, Thomas/ Bidlingmeyer, Brian.Analysis of amino acids using precolumn derivatization with phenylisothiocyanate.American  Laboratory Aug 1984. -PITC

    D'Aniello, Antimo/ Petrucelli, Leonard/ Gardner, Christina/ Fisher, George.  Improved Method for Hydrolyzing Proteins and Peptides without Inducing Racemization and for Determining Their True D-Amino Acid Content.  Analytical Biochemistry 213, 290-295 (1993)

    Hancock, Diane/ Reeder, Dennis.  Analysis and configuration assignments of the amino acids in a pyroverdine-type siderophore by reversed-phase high-performance liquid chromatography.  Journal of Chromatography, 646 (1993) 335-343.  -PITC

    Hariharan, M./ Naga, Sundar/ VanNoord, Ted. Systematic approach to the development of plasma amino acid analysis by high-performance liquid chromatography with ultraviolet detection with precolumn derivatization using phenyl isothiocyanate.  Journal of Chromatography, 621 (1993) pgs. 15-22. -PITC

    Janssen, P./ van Nispen,J./ Melgers, P./ van den Bogaart, H./ Hamelinck, R./ Goverde, B. HPLC Analysis of Phenylthiocarbamyl (PTC) Amino Acids. I. Application in the Analysis of (Poly)peptides.  Chromatographia Vol. 22, No. &-12, pg. 351-357, Dec. 1986. -PITC

    Jonge, Leon/ Breuer, Michel.  Modification of the analysis of amino acids in pig plasma.  Journal of Chromatography B, 6542 (1994) 90-96. 

    Kawasaki , Takao/ Higuchi, Takeru/ Imai, Kazuhiro/ Wong, Osborne.  Determination of Dopamine, Norepinephrine, and Related Trace Amines by Prochromatographic Derivatization with Naphthalene-2,3-dicarboxaldehyde. Analytical Biochemistry 180, 279-285 (1989) -NDA-CN

    Kemp, B.E. (1980) Relative alkali stability of some peptide o-phosphoserine and o-phosphothreonine esters.  FEBS Lett.110, 308-312.

    LeFevre, Joseph.  Reversed-phase thin-layer chromatographic separations of enantiomers of dansyl-amino acids using B-cyclodextrin as a moblie phase additive.  Journal of Chromatography A, 653 (1993) 293-302

    Liu, T.-Y. and Chang, Y.H. (1971) Hydrolysis of proteins with p-toluenesulphonic acid. Determination of tryptophan. J. Biol. Chem.  246, 2842-2848.

    Lobell, Mario/ Schneider, Manfred.  2,3,4,6-Tetra-O-benzoyl-B-D-glucopyranosyl isothiocyanate:an efficient reagent for the determination of enantiomeric purities of amino acids, B-adrenergic blockers and alkyloxiranes by high-performance liquid chromatography using standard reversed-phase columns.  Journal of Chromatography, 633 (1993) 287-294. AGIT

    Lunte, Susan. Naphthalenedialdehyde-cyanide:A versatile fluorogenic reagent for the LC analysis of peptides and other primary amines.  >LC-GC Vol. 7, No. 11 pg. 908-916 -NDA-CN

    Lunte, Susan/ Mohabbat, Tariq/ Wong, Osborne/ Kuwana, Theodore. Determinatin of Desmosine Idodesmosine, and Other Amino Acids by Liquid Chromatography with Electrochemical Detection following Precolumn Derivatization with Naphthalenedialedhyde/Cyanide.  Analytical Biochemistry 178, 202-207 (1989) -NDA

    Martensen, T.M.  (1982) Phosphotyrosine in proteins.  Stability  and quantification.  J. Biol. Chem. 257, 9648-9652.

    Matsubara & Sasaki, BBRC 35, 175-181 (1969).

    Matuszewski, Bogdan/ Givens, Richards/ Srinivasachar, Kasturi/ Carlson, Robert/ Higuchi, Takeru.  N-Substituted 1-Cyanobenz[f]isoindole: Evaluation of Fluoresence Efficiencies of a New Fluorogenic Label for Primary Amines and Amino Acids.  Analytical Chemistry, Vol. 59, Page 1102, (1987) -NDA-CN

    Montigny, Pierre/ Stobaugh, John/ Givens, Richard/ Carlson, Robert/ Srinivasachar, Kasturi/ Sternson, Larry/ Higuchi, Takeru.  Naphthalene-2,3-dicarboxaldehyde/Cyanide Ion: A Rationally Designed Fluorogenic Reagent for Primary Amines.  Analytical Chemistry (1987) Vol. 59, pg. 1096. -NDA-CN

    Moore, S. and Stein, W.H. (1951) J. Biol. Chem. 178, 53-77.

    Moore, S. and Stein, W.H. (1954) J. Biol. Chem. 211, 893-906.

    Moore, S. and Stein, W.H. (1963) Methods Enzymol. 6, 819-831 (1963).

    Neidle, Amos/ Banay-Schwartz, Miriam/ Sacks, Shirley/ Dunlop, David.  Amino Acid Analysis Using 1-Napthylisocyanate as a Precolumn High Performance Liquid Chroamatography Derivatization Reagent.  Analytical Biochemistry 189 (1989) 291-297. -PITC/ OPA/ FMOC-Cl

    Nimura, N., Iwaki, K., Kinoshita, T., Takeda, K. and Ogura, H. (1986) Anal. Chem. 58, 2372-2375.

    Organon, Janssen.  PTC-Amino Acid separation: C18 column.  Chromatographia, 22: 345-357 (1986) -PTC

    Penke et al., Anal. Biochem. 60, 45-50 (1974).

    Roach, Marc/ Harmony, Marlin.  Determination of Amino Acids at Subfemtomole Levels by High-Performance Liquid Chromatography with Laser-Induced Fluorescence Detection.  Analytical Chemistry, 1987, vol. 59 pg. 411.  -OPA

    Slater, George/ Manville, John.  Analysis of thiocyanates and isothiocyanates by ammonia chemical ionization gas chromatography-mass spectrometry and gas chromatography-Fourier transform infrared spectroscopy.  Journal of Chromatography, 648 (1993) 433-443 -ITC

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    Strydom, D., Tarr, G., Pan, Y-C and Paxton, R., in  Techniques in Protein Chemistry II (R.H. Angeletti, ed.), Academic Press, (1992) San Diego , pp. 261-274.

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    <div><b><font size="4">Abbreviations</font></b></div> <table> <tbody> <tr> <td width="50">Ar</td> <td>Arabidopsis</td> </tr> <tr> <td>B</td> <td>Bovine</td> </tr> <tr> <td>BA</td> <td>Baboon</td> </tr> <tr> <td>Bc</td> <td>Bacteria</td> </tr> <tr> <td>Bl</td> <td>Blocking</td> </tr> <tr> <td>Bt</td> <td>Biotinylated</td> </tr> <tr> <td>Ce</td> <td>C. elegans</td> </tr> <tr> <td>C</td> <td>Chicken</td> </tr> <tr> <td>Chm</td> <td>Chimpanzee</td> </tr> <tr> <td>CP</td> <td>Carp</td> </tr> <tr> <td>CT</td> <td>C-Terminus</td> </tr> <tr> <td>D</td> <td>Dog</td> </tr> <tr> <td>Ds</td> <td>Drosophila</td> </tr> <tr> <td>E</td> <td>ELISA</td> </tr> <tr> <td>ED</td> <td>Extracellular Domain</td> </tr> <tr> <td>EL</td> <td>Extracellular Loop</td> </tr> <tr> <td>F</td> <td>Feline</td> </tr> <tr> <td>FACS</td> <td>Flow Cytometry</td> </tr> <tr> <td>Fu</td> <td>Fungus</td> </tr> <tr> <td>G</td> <td>Guinea Pig</td> </tr> <tr> <td>Gt</td> <td>Goat</td> </tr> <tr> <td>H</td> <td>Human</td> </tr> <tr> <td>HM</td> <td>Hamster</td> </tr> <tr> <td>Hr</td> <td>Horse</td> </tr> <tr> <td>I</td> <td>Immunogen</td> </tr> <tr> <td>ICC</td> <td>Immunocytochemistry</td> </tr> <tr> <td>ID</td> <td>Intracellular Domain</td> </tr> <tr> <td>IF</td> <td>Immunofluorescence</td> </tr> <tr> <td>IHC</td> <td>Immunohistochemistry</td> </tr> <tr> <td>IN</td> <td>Intermediate Domain</td> </tr> <tr> <td>IP</td> <td>Immunoprecipitation</td> </tr> <tr> <td>M</td> <td>Mouse</td> </tr> <tr> <td>Ma</td> <td>Mammal</td> </tr> <tr> <td>Mk</td> <td>Monkey</td> </tr> <tr> <td>MS</td> <td>Mass Spectrometry</td> </tr> <tr> <td>Mt</td> <td>M. tuberculosis</td> </tr> <tr> <td>N</td> <td>Neutralization</td> </tr> <tr> <td>NT</td> <td>N-Terminus</td> </tr> <tr> <td>P</td> <td>Porcine</td> </tr> <tr> <td>Pa</td> <td>Parasite</td> </tr> <tr> <td>PF</td> <td>Puffer Fish</td> </tr> <tr> <td>Pl</td> <td>Plant</td> </tr> <tr> <td>Pz</td> <td>Protozoan</td> </tr> <tr> <td>R</td> <td>Rat</td> </tr> <tr> <td>Rb</td> <td>Rabbit</td> </tr> <tr> <td>S</td> <td>Sheep</td> </tr> <tr> <td>V</td> <td>Virus</td> </tr> <tr> <td>WB</td> <td>Western Blot</td> </tr> <tr> <td>X</td> <td>Xenopus</td> </tr> <tr> <td>Y</td> <td>Yeast</td> </tr> <tr> <td>ZF</td> <td>Zebra Fish</td> </tr> </tbody> </table> <br />

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    Abbreviations
    Ar Arabidopsis
    B Bovine
    BA Baboon
    Bc Bacteria
    Bl Blocking
    Bt Biotinylated
    Ce C. elegans
    C Chicken
    Chm Chimpanzee
    CP Carp
    CT C-Terminus
    D Dog
    Ds Drosophila
    E ELISA
    ED Extracellular Domain
    EL Extracellular Loop
    F Feline
    FACS Flow Cytometry
    Fu Fungus
    G Guinea Pig
    Gt Goat
    H Human
    HM Hamster
    Hr Horse
    I Immunogen
    ICC Immunocytochemistry
    ID Intracellular Domain
    IF Immunofluorescence
    IHC Immunohistochemistry
    IN Intermediate Domain
    IP Immunoprecipitation
    M Mouse
    Ma Mammal
    Mk Monkey
    MS Mass Spectrometry
    Mt M. tuberculosis
    N Neutralization
    NT N-Terminus
    P Porcine
    Pa Parasite
    PF Puffer Fish
    Pl Plant
    Pz Protozoan
    R Rat
    Rb Rabbit
    S Sheep
    V Virus
    WB Western Blot
    X Xenopus
    Y Yeast
    ZF Zebra Fish


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    Bridged Nucleic Acids (BNAs) - BNA3

    Beyond peptide nucleic acids (PNAs) and locked nucleic acids (LNAs)

    The near completion of the human genome sequence in 2001 as well as the availability of whole genome sequences for many organisms in recent years has led to an exponential increase in genomic information in the last decade. The NIH website, http://www.ncbi.nlm.nih.gov/genome, contains a collection of sequences for partial or whole genomes, the number of which is constantly increasing at a high speed. This huge amount of sequence information provides a large source for the design of genetic tools to study molecular details of the genetic information flow in organisms. Scientific evidence shows that humans and other vertebrates are made up by close to or more than 220 different specialized cell types. Each cell type has its own gene regulatory network. To understand an organism at the molecular level the regulation of the genetic information flow will need to be studied. For this purpose new tools have been and are constantly developed. The double helix of DNA is nature’s solution to storing, retrieving, and communicating genetic information of a living organism. Two of many important characteristics of the DNA molecule are the specificity and the reversible nature of the hydrogen bonding between complementary nucleobases. These properties allow the DNA strands of the double helix to unwind and rewind in exactly the same configuration. It is now understood that the cell contains a vast collection of ‘nano-machines’ that control gene regulation. DNA, RNA, proteins, and other molecules make up these nano-assemblies. Control points for gene expression that are studied by scientists include transcriptional regulation, RNA processing, translational control, the stability of mRNA, posttranslational control, and DNA rearrangements among others. These regulatory systems found in prokaryotes and eukaryotes can differ from each other in many details. Once the nature of the DNA and RNA molecules was established the field of life science realized early on that if specific, single strands of DNA could be synthesized, scientist would be able to study and manipulate the gene sequences of DNA and RNA. This was realized with the development of efficient chemistries including automated instrumentation to allow for the synthesis of DNA and RNA monomers and polymers of increased size including their modifications. Oligonucleotide chemistry has been developed greatly over the past three decades and many advances have been made in the design of DNA, RNA and peptide based molecular tools with increased nuclease resistance.

     

    The double helix exists in multiple conformations which were revealed by X-ray diffraction studies of concentrated DNA solutions that had been drawn out into thin fibers. Two kinds of structures, the B and the A forms of DNA were found. It is thought that the B form corresponds to the average structure of DNA under physiological conditions. Its structure has 10 base pairs per turn, and a wide major groove and a narrow minor groove. The A form on the other hand has 11 base pairs per turn and its major groove is narrower and much deeper than that of the B form. Furthermore, its minor groove is broader and shallower. The B DNA is found in the vast majority of the DNA in the cell. However, A DNA can be found in certain DNA-protein complexes. The non-covalent bonds in double stranded DNA (dsDNA) sequences are formed by Watson-Crick interactions. On the other hand, in triplex forming oligonucleotides the triplex forming strand binds to the duplex via Hoogsteen hydrogen interactions. Structures of the nucleotides and their interaction are shown in the following figure.


    Structures of Watson-Crick-type base pairs, G•C and A•T (left), and Hoogsteen type base triads, C+•G•C and T•A•T (right).

    The chemical structures for RNA and DNA are shown below as well as the structures for the A form of RNA and the B-form of DNA.

    RNA contains ribose rather than 2’-deoxyribose in its backbone. The ribose has a hydroxyl group at the 2’-position. Furthermore, RNA contains the nucleic acid uracil in place of thymine and is usually found as a single polynucleotide chain. While RNA is typically single stranded, RNA chains can frequently fold back on themselves to form base-paired segments between short stretches of complementary sequences. The presence of 2’-hydroxyls in the RNA backbone favors a structure that resembles the A-form structure of DNA. The flexible five-membered furanose ring in nucleotides exists in equilibrium of two conformations of the N- and the S-type. A closer look at the two forms is shown as follows.

    Synthetic oligonucleotides are now important, established tools for life scientists and have many applications in molecular biology, genetic diagnostics and are poised to become important tools in the emerging field of molecular medicine as well. While unmodified oligodeoxynucleotides can form DNA:DNA and DNA:RNA duplexes they are sometimes unstable and labile to nucleases. Therefore a variety of nucleic acid analogs have been developed to enhance high-affinity recognition of DNA and RNA targets, enhancing duplex stability and assist with cellular uptake. Among the list of these analogs are peptide nucleic acids (PNAs), 2’-fluoro N3-P5-phosphoamidites, 1’, 5’-anhydrohexitol nucleic acids (HNAs) and locked nucleic acids (LNAs) as well as other bridged nucleic acids (BNAs). Peptide nucleic acids (PNAs) are synthetic polymers that contain a peptide backbone and nucleic acid bases as side chains within their sequence. These polymers can form strong specific bonds with complementary sequences present in double-stranded DNA (dsDNA). 2’-fluoro N3-P5-phosphoamidites are modified nucleotide analogs that contain fluor in the 2’ position. Hexitol nucleic acids are oligonucleotides built up from natural nucleobases and a phosphorylated 1,5-anhydrohexitol backbone. Anhydrohexitol oligonucleotides can be synthesized using phosphoramidite chemistry and standard protecting groups. LNAs are structurally rigid oligo-nucleotides with increased binding affinities.

     

    A bridged nucleic acid (BNA) is a molecule that can contain a five-membered or six-membered bridged structure. Ideally the bridge is synthetically incorporated at the 2’, 4’-position of the ribose to afford a 2’, 4’-BNA monomer. The monomers can be incorporated into the oligonucleotide polymer structure using standard phosphoamidite chemistry. The goal for the synthesis and use of BNAs is to generate oligonucleotides with (i) equal or higher binding affinity against an RNA complement with excellent single-mismatch discriminating power, (ii) much better RNA selective binding, (iii) stronger and more sequence selective triplex-forming characters, and (iv) with a pronounced higher nuclease resistance, even higher than Sp-phosphorthioate analogues, than regular DNA or RNA oligonucleotides.


    Monomer structures of BNAs. The structures of selected nucleic acid analogs with a bridged sugar moiety are depicted.
     

    While a large number of chemically modified oligonucleotides have been developed during the last few decades, most of these molecules have failed to give the desired response and the search for new molecules with better qualities still continues today. One dramatic improvement was made with the introduction of the bridged nucleic acid, 2’, 4’-BNA (also called LNA). The compound shows a better hybridization affinity for complementary strands, both for RNA and DNA strands, in comparison to unmodified nucleotides. Furthermore the BNA can be used to design sequence selective LNA-oligonucleotide hybrids that are soluble in aqueous solutions and exhibit improved biostability in comparison to natural nucleotides. This BNA monomer has now been widely used in nucleic-acid-based technologies. However, according to Imanishi’s group, there is a need for further development because the nuclease resistance of the LNA is significantly lower than that obtained by phosphorothioate oligonucleotide and the fact that oligonucleotides containing consecutive LNA units or a fully modified oligonucleotide using the analog are very rigid and inflexible. Furthermore, additional research has proven that a kind of LNA-modified antisense oligonucleotide is hepatotoxic.

     

    The scientists Satoshi Obika, Daishu Nanbu, Yoshiyuki Hari, Ken-ichiro Morio, Yasuko In, Toshimasa Ishida, and Takeshi Imanishi in 1997 reported the synthesis of a 2’-O,4’-C-Methyleneneuridine and –cytidine, novel bicylic nucleosides with a fixed C3 - endo sugar puckering. One of these nucleoside analogs is now known as a “locked nucleotide” or LNA. In the ribofuranose, the plane C1’-O4’-C4’ is fixed. The C3’- endo conformation is found in RNA. DNA can adjust and is able to take on both conformations. The exact nomenclature of sugar puckers can be found at http://www.chem.qmul.ac.uk/iupac/misc/pnuc2.html

     

    Obika et al. in 1998 (Imanishi’s research group) report the synthesis of bicyclic nucleoside analogues with a fixed N-type conformation, 2'-O,4'-C-methyleneuridine and –cytidine and the incorporated of this analogue into oligonucleotides. The binding efficiency of the modified oligonucleotides to the complementary DNA and RNA as well as the CD spectra of the modified DNA-DNA and modified DNA-RNA duplexes were studied.

     

    Imanishi’s group has synthesized newer generations of BNAs with improved properties, one called 2’4’-BNA-COC, and more recently, one called 2’,4’-BNA-NC. To optimize the length of the bridging moiety the BNA-NC was designed to contain a six-membered bridged structure with an N-O bond in the sugar molecule. The next figures show the chemical structures and 3D ball-and-stick model for the three BNAs.


    The chemical structures and the energy minimized molecular models for the 2’, 4’-BNA-NC[NH] and 2’, 4’-BNA-NC[NCH3] monomers are illustrated here.
     

    Hari et al. in 2003 developed a novel nucleoside analogue for the effective recognition of CG interruption in a homopurine–homopyrimidine tract of double-stranded DNA (dsDNA). The group succeeded to synthesize a triplex-forming oligonucleotide (TFO) containing a novel 2’,4’-BNA (QB) bearing 1-isoquinolone as a nucleobase. The group used this BNA to investigate the triplex-forming ability and sequence-selectivity of the TFO. Using melting temperature (TM) measurements, it was found that the TFO-QB formed a stable triplex DNA in a highly sequence-selective manner under near physiological conditions.

     

    Rahman et al. reported in 2007 that oligonucleotides containing 2’, 4’-BNA-NC monomers show an increase in their melting temperature (Tm) of 5.3 to 6.3 °C per modification (ΔT/mod) when investigated by UV melting experiments (Tm measurements).The melting temperature (Tm) is defined as the temperature at which half of the DNA strands are in the double-helical state and half are in the random coil state. The Tm depends on the length of the oligonucleotide molecule and its sequence. The Tm values of duplexes formed by 2’, 4’-BNA-NC[NH] oligonucleotides that contained three or more modifications are higher than those exhibited by the corresponding 2’, 4’-LNA modified oligonucleotides indicating a stronger binding and stability of the duplexes. Both 2’, 4’-BNA-NC[NH] and 2’, 4’-BNA-NC[NCH3] display selective binding to RNA, superior to that of 2’, 4’-LNAs. The researchers used mismatch discrimination studies to evaluate their ability for selective hybridization. The presence of a mismatched base in the target RNA strand resulted in a substantial decrease in the Tm of the duplexes formed. The analysis of formed duplexes by circular dichroism (CD) verified the presence of the A-form by showing the spectra typical for this structure. The A-form of the helical structure is favored because of the conformational restriction of the sugar moiety to the N-form. Furthermore, the BNAs can form triplexes with a higher stability compared to that of LNAs. Applications in antigenic and gene repair technologies require the formation of stable triplexes at physiological pH. Next, to test the BNAs for their resistance to nucleases oligonucleotides [5’-d(TTTTTTTTTT)-3’] modified with a single 2, 4’-BNA-NC unit were incubated with a 3’-exonuclease (Crotalus adamanteus venom phosphodiesterase, CAVP, Pharmacia) at 37 °C and the amount of intact oligonucleotides remaining were evaluated by RP-HPLC. The results showed that oligonucleotides modified with BNA-NC[NMe] are much more resistant to degradation than LNA modified oligonucleotides.

     

    Rahman et al. in 2007 report on the use of BNA analogues to allow the formation of highly stable pyrimidine-motif triplexes at physiological pH. The formation of a stable triplex DNA molecule at physiological pH values is a highly desirable phenomenon in molecular biology and medicinal chemistry because of its function in the regulation of gene expression, site-specific cleavage of DNA, gene mapping and isolation, the maintenance of folded chromosome conformations, and gene-targeted mutagenesis. The researchers argue that in a pyrimidine-motif triplex DNA, the (homopyrimidine) triplex-forming oligonucleotide (TFO) binds to the homopurine tract of the target duplex DNA. This binding is sequence specific and maintained through Hoogsteen hydrogen bonds to form T·A:T and C+·G:C triads. The formation of the C+·G:C triad depends on the protonation of cytosine, which is only favorable at acidic pH values (pKa=4.5). Therefore, homo-pyrimidine-motif triplexes are extremely unstable at physiological pH values severely limiting their biological application. The group synthesized a novel bridged nucleic acid analogue, 2’,4’-BNANC, and demonstrated that the TFOs composed of 2’,4’-BNANC formed highly stable pyrimidine-motif triplexes at physiological pH values. Furthermore they show that the TFOs eliminate the requirement of placing alternating DNA monomers for optimum efficacy needed when using LNAs. They could show that fully modified TFOs still formed a highly stable triplex. They stated that “these promising properties of 2’,4’-BNANC will be helpful for developing oligonucleotide-based technologies for the postgenome era.”

     

    Also in 2007, Imanishi’s group synthesized oligonucleotides that were modified with a novel BNA analogue, 2’, 4’-BNANC[N–CH3], and compared their properties to oligonucleotides containing 2’,4’-BNA (LNA). They showed that these oligonucleotides have a similarly high RNA affinity but a better selectivity for RNA and a much higher resistance to nuclease degradation. These results suggested that the novel BNA analogue may be particularly useful for antisense approaches when used for the design of antisense oligonucleotides.

     

    Kasahara et al. in 2010 published a paper in where they showed that the capping of the 3’-ends of thrombin binding aptamers (TBAs) with bridged nucleotides increased the nuclease resistances and the stability of the aptamers in human serum. The capping did not affect the binding abilities of the aptamers. The researchers report that the capping could be achieved via a one step enzymatic process using 2’, 4’-bridged nucleoside 5’-triphosphate and the enzyme terminal deoxynucleotidyl transferase.

     

    In the same year Rahman et al. reported the results of their study in which a number of 2’,4’-BNA- and 2’,4’-BNANC-modified siRNAs were designed and synthesized. The thermal stability, nuclease resistance and gene silencing properties against cultured mammalian cells were evaluated and compared with those of natural siRNAs. The 2’,4’-BNA- and 2’,4’-BNANC-modified siRNAs showed very high TM values and were remarkably stable in serum samples. Furthermore, the researchers report that these modified oligo-nucleotides showed promising RNAi properties that were equal to those exhibited by natural siRNAs. The thermally stable siBNAs composed of slightly modified sense and antisense strands suppressed gene expression equal to that of natural siRNA. The modifications at the Argonaut (Ago2) cleavage site of the sense strand (9–11th positions from the 5’-end of the sense strand) produced variable results depending on siRNA composition. However, modification at the 10th position diminished siRNA activity. In moderately modified siRNAs, modification at the 11th position displayed usual RNAi activity, while modification at the 9th position showed variable results depending on the composition of the siRNA.

     

    Yamamoto et al. in 2012 demonstrated that BNA-based antisense therapeutics can be used to successfully inhibit hepatic PCSK9 expression which resulted in a strong reduction of the serum LDL-C levels of mice. These findings support the hypothesis that PCSK9 is a potential therapeutic target for hypercholesterolemia. The researchers state that this is the first time they were able to show that BNA-based antisense oligo-nucleotides (AONs) induced a cholesterol-lowering action in hypercholesterolemic mice. Hypercholesterol-emia is a metabolic condition in which high levels of cholesterol are present in the blood and where elevated levels of lipids and lipoproteins are observed in the blood as well. If untreated higher levels of total cholesterol will increase the risk for cardiovascular disease in particular coronary heart disease.

     

    We expect that BNAs like LNAs will find a widespread use in antisense oligonucleotide technology, where they can be used to stabilize interactions with target RNA and protect them from the attack by cellular nucleases. Furthermore, they can be used in the field of molecular diagnostics and the newly emerging field of siRNAs. Utilizing these modified nucleotides promises to increase double-stranded RNA stability in serum and decrease off-target effects seen with conventional siRNAs. Next, this oligonucleotide technology has the potential to become a new type of therapy to treat a wide variety of diseases, and BNAs will no doubt play a part in future developments of therapeutic and diagnostic oligonucleotides.

    Comparison of various nucleic acid analogs

    Criteria BNA LNA PNA DNA
    Hybridization affinity with DNA 1-3 °C higher Tm per base 2-3 °C higher Tm per base At least 1 ℃ higher Tm per base -
    Salt concentration for hybridization Dependent Dependent Independent Dependent
    Tm for each single mismatch Ca. 4 ℃ Ca. 3-4 ℃ Lowering 1 - 5℃ Lowering 1℃
    Chemical stability Stable to moderately stable Stable to moderately stable Stable Unstable or moderately stable
    Biological stability Very stable Stable Stable to nuclease and protease Degradation by nuclease
    Thermal stability Excellent Good Good Moderate
    Water solubility Excellent Good to Excellent Soluble Soluble
    Probe length for diagnostic use 10-25 10-25 13 - 18 bases 20-30 bases
    PCR compatible Yes Yes No Yes
    Ability to introduce other nucleic acids in oligonucleotide Yes Yes No Yes
    Body clearance ability Yes Yes No Yes
    INhibition of RNAse H Yes Less No Yes
    Triplex formation Yes Yes Yes Yes
    Hepatotoxicity No Moderate Moderate No
    Nephrotoxicity No very low High No
    Innate immunity stimulation No No Yes Yes
    Gene Silencing Yes Yes Yes na
    Triplex Formation Ability Excellent Moderate Good Yes
    RNA/DNA binding selectivity Excellent Good No preference na
    Hybridization affinity with RNA 5-6 °C higher Tm per base 5-6 °C higher Tm per base At least 1 °C higher Tm per base na
    Nuclease Resistance Yes Yes Yes No
    SNP detection Yes Yes Yes na
    Quantitativereal-time PCR applications Yes Yes Yes na
    genotyping experiments Yes Yes Yes na
    Telomere FISH Probes Yes Yes Yes na
    Other FISH Probes Yes Yes Yes na
    Design of shorter fluorescent probes Excellent Good Good no
    Improved mismatch discrimination. Excellent Good Good no
    Increases the window of annealing temperatures for accurate genotyping Excellent Good Good no
    Various formats of detection Excellent Good Good no
    Dection with dual-labeled probes, Excellent Good Good no

    Rules for the Design of Probes

    LNAs # BNAs PNAs
    (i) Place a triplet of LNA modifications with the central base of the triplet at the mismatch site, unless the probe contains the guanine base of a G_T mismatch. (i) One or more consecutive BNANC monomer(s) within an oligonucleotide may be replaced. This works well for diagnostic applications. The mode of BNANC depends on the application. For example, both gapmer- and chimera-type modifications using BNANC will work well for antisense applications. To recruit RNase H the incorporation of more than four (4) consecutive BNAs are needed.
    Recommended sequence length => 10 to 18 bases, not including linkers, amino acids and labels. 1 Lys or Cys can be attached to either the N-terminus or the C-terminus.
     
    Purine Content:Purine-rich PNA oligomers tend to aggregate and have low solubility in aqueous solutions. To avoid aggregation, follow the following guidelines:
     
    (i) Limit the purine content to 60%.
    Example: GAT TAG CAG TCT ACG
    (Acceptable - Purine content < 60%)
    (ii) LNA modification of the guanine nucleotide or either of its nearest-neighbor bases should be avoided in a G_T mismatch site. (ii) Several BNANC monomers can be placed next to each other within the internal nucleotide stretch depending on selected application. (ii) The maximum purine-stretch = 4 in a row, and the maximum stretch of guanines = 3-in-a-row
    Examples: ATT AGG GGC ATC TAC (Not acceptable - 4 G's in a row)
    CTA GAT AGA AGG TTC (Not Acceptable - 6 purines in a row)
    (iii) Shorter probes improve mismatch discrimination. (iii) In some cases it is best not to place BNA monomers at the ends of the oligonucleotide (5'/3') within 3 nucleotides. However, BNANC monomers can be used at the ends as well.  Generally shorter probes improve mismatch discrimination.
    (iii) Consider probing the complementary strand if you violate the above guidelines.
    Examples: GTA GAT GCC CCT AAT (Acceptable -Complement of the above sequence does not violate any of the guidelines)
    GAA CCT TCT ATC TAG (Acceptable -Complement of the above sequence)
    (iv) For best synthesis quality, it is preferable to avoid consecutive sequence of more than four LNA residues.
    (iv)  The same type of BNA monomers can be used within the oligo-nucleotide sequence selected. For example, more than four (4) consecutive BNAs are needed to recruit RNase H.
    iv) Avoid self-complementary sequences with inverse repeats, hairpins and palindromes. These types of probes are prone to aggregation, Since PNA/PNA interactions are even stronger than PNA/DNA interactions.
    Four base complements are acceptable, unless they contain only C and G. However CG complements are acceptable when interrupted by one or more bases.
    Examples: GAT AATT GCA (Acceptable - four bases complement)
    GAT CCGG TAC (Not acceptable - CCGG complement)
    TAT CCT GGT A (Acceptable CC, GG interrupted by a base)
    (v) Better discrimination seems to be achieved when the position of the mismatch site is close to the center of the probe. Discrimination is significantly decreased if mismatches are located at the first or second base from either end of the duplex. (v) Generally the use of no more than 4-8 BNA’s within a 20 mer probe is recommended. However, a higher or lower modification rate is possible.  The design is based on teh application.
    (v) Complements that are 6 bases or more in length are unacceptable.
    Example:
    CTA TTA ATG CA (Not acceptable - six bases complement)
    (vi) If a mismatch type and location are unknown, the best approach is to modify all adenine nucleotides until the optimal Tm is achieved. The rest of the rules should be taken into account.
    (vi) Each BNANC monomer increase the Tm by about 4 degrees Celsius. Therefore this can be used to estimate the Tm of the BNA containing oligonucleotide).
    (vi) Complements of 6 bases are acceptable when interrupted by one or more bases, unless only C and G are used.
    Example:
    AGT GCT ACT (Acceptable - interrupted six bases complement )
    GCG GCT CGC (Not acceptable - G and C only complement
    (vii) Probes should not fold into stable, undesired selfcomplementary secondary structures or form self-dimers, especially when these structures contain LNA–LNA base pairs. (vii) An oligonucleotide designed with 2’, 4’-BNA-NC[NH] is more selective to ssRNA and binds more strongly than LNA if more bases are modified.
    (vii) Complements of 8 bases are unacceptable even when interrupted by one or more bases.
    Example:
    ACTG T CAGT (Not acceptable - eight bases complement)
    Rules (iii) and (iv) should be balanced depending on the precise needs of specific applications. Fully modified short LNA probes (6–8 bp) have been used successfully in SNP detection experiments. (viii) An oligonucleotide designed with 2’, 4’-BNANC[NH] binds also to ssDNA and binds also more strongly than LNA if more bases are modified. The ssDNA binding strength of BNANC(NH) is equivalent to or slightly more than that of LNA. Note 1: PNAs can form duplexes in either orientation, but an anti-parallel orientation is strongly preferred and will form the most regular duplex. The anti-parallel configuration is preferred for antisense and DNA probe-type applications. When the orientation of the PNA is anti-parallel, the N-terminal of the PNA probe is equivalent to the 5'-end of the DNA.
    Less suitable for some high-throughput screenings where each experiment cannot be thoroughly optimized, and varying synthesis yields as well as extensive purifications are incompatible. Longer LNAs (>14 bp) may possess too high affinity for complementary strands when they are fully modified (viiii) Modification with the BNANC (NCH3) monomer dramatically increase nuclease-resistant (much more than found in LNA modified oligonucleotides).  This property is desirable not only for therapeutic application but also for diagnostic use. Note 2: The PNA strand is uncharged, a PNA•DNA-duplex will have a higher Tm than the corresponding DNA-DNA-duplex. Typically there will be an increase in Tm of about 1°C per base pair at 100 mM NaCl. At lower salt concentrations the Tm differences will be even more dramatic. A 10-mer PNA will typically have a Tm of about 50°C, and a 15-mer PNA will typically have a Tm of 70° C.

    Additional experimentation is needed to obtain more insight about advantages and disadvantages of short fully LNA-modified probes versus chimeric LNA/DNA probes.

    IDT SciTools OligoAnalyzer calculator freely accessible at www.idtdna.com

    Please review the basic properties and characteristics for the application of BNANC in the following references:
     
    Chem. Commun., 2007, 3765;
    Angew. Chem. Int. Ed., 2007, 46, 4306;
    J. Am. Chem. Soc., 2008, 130, 4886; Bioorg. Med. Chem., 2010, 18, 3473; Molecular Therapy NA, 2011, 1, e22;
    J. Nucleic Acids, 2012, ID 707323.
    A. Sequence length is primarily determined by the required specificity of the application. DNA applications that require more than 25 bases can be routinely performed with much shorter PNA probes. Long PNA oligomers, depending on the sequence, tend to aggregate and are difficult to purify and characterize. However, the shorter a sequence is, the more specific it is. Consequently, the impact of mismatch is greater for a short sequence.
    # You et al. 2006 proposed the following rules to maximize single mismatch discrimination using LNA probes.

    Benefits of the BNA technology include:

    * Ideal for the detection of short RNA and DNA targets
    * Increases the thermal stability of duplexes
    * Increases the thermal stability of triplexes
    * Capable of single nucleotide discrimination
    * Resistant to exo- and endonucleases resulting in high stability in vivo and in vitro applications
    * Increased target specificity
    * Facilitates Tm normalization
    * Strand invasion properties enable the detection of “hard to access” samples
    * Compatible with standard enzymatic processes
    Applications

    • Design and synthesis of RNAaptamers,
    • siRNA,
    • antisense probes,
    • diagnostics,
    • Isolation
    • Microarray analysis
    • Northern blotting
    • Real-time PCR
    • In situ Hybridization
    • Functional analysis
    • SNP detection
    • to use as antigens and many others nucleotide base applications.
    Figures below show graphical illustrations of the function and use of BNAs.

    References

    Braasch DA, and DR Corey 2001 Locked nucleic acid (LNA): fine-tuning the recognition of DNMA and RNA. Chem Biol 8: 1-7.

    Braasch DA, Y Liu, and DR Corey 2002 Antisense inhibition of gene expression in cells by oligonucleotides incorporating locked nucleic acids: effect of mRNA target sequence and chimera design. NAR 30: 5160-5167.
     
    Grunweller A, E Wyszko, B Bieber,R Jahnel, VA Erdmann, and J Kurreck 2003, Comparison of different antisense strategies in mammalian cells using locked nucleic acids, 2’-O-methyl RNA, phosphorothioates and small interfering RNA. NAR 31: 3185-3193.

    Hendrix C, H Rosemeyer, B De Bouvere, A Van Aerschot, F Seela, and P Herdewijn 1997 1',5'-Anhydrohexitol oligonucleotides: hybridisation and strand displacement with oligoribonucleotides, interaction with RNase H and HIV reverse transcriptase. Eur J Chem 3: 1513–1520.

    Yoshiyuki Hari, Satoshi Obika, Mitsuaki Sekiguchi and Takeshi Imanishi; Selective recognition of CG interruption by 20,40-BNA having 1-isoquinolone as a nucleobase in a pyrimidine motif triplex formation. Tetrahedron 59 (2003) 5123–5128.

    Hyrup B, and PE Nielson 1996 Peptide nucleic acids (PNA): synthesis, properties and potential applications. Bioorg Med Chem 4:5-23.

    Takeshi Imanishi and Satoshi Obika; BNAs: novel nucleic acid analogs with a bridged sugar moiety. CHEM. COMMUN., 2002, 1653–1659.

    Johnson MP, LM Haupt, and LR Griffiths 2004 Locked nucleic acids (LNA) single nucleotide polymorphism (SNP) genotype analysis and validation using real-time PCR. NAR 32: e55.

    Yuuya Kasahara, Shunsuke Kitadume, Kunihiko Morihiro, Masayasu Kuwahara, Hiroaki Ozaki, Hiroaki Sawai, Takeshi Imanishi, Satoshi Obika; Effect of 3’-end capping of aptamer with various 2’,4’-bridged nucleotides: Enzymatic post-modification toward a practical use of polyclonal aptamers. Bioorganic & Medicinal Chemistry Letters 20 (2010) 1626–1629.

    Harleen Kaur, B. Ravindra Babu, and Souvik Maiti; Perspectives on Chemistry and Therapeutic Applications of Locked Nucleic Acid (LNA). Chem. Rev. 2007, 107, 4672-4697.

    Tetsuya Kodama,Chieko Matsuo, Hidetsugu Ori, Tetsuya Miyoshi, Satoshi Obika, Kazuyuki Miyashita, Takeshi Imanishi; Design, synthesis, and evaluation of a novel bridged nucleic acid, 2’,5’-BNAON, with S-type sugar conformation fixed by N–O linkage. Tetrahedron 65(2009) 2116–2123.

    Koshkin AA, SK Singh, P Nielsen, VK Rajwanshi, R Kumar, M Meldgaard, CE Olsen, and J Wengel 1998 LNA (Locked Nucelic Acid): Synthesis of the adenine, cytosine, guanine, 5-methylcytosine, thymine and uracil bicyclonucleoside monomers, oligomerisation, and unprecedented nucleic acid recognition. Tetrahedron 54: 3607-3630.

    Kurreck J, E Wyszko, C Gillen, and VA Erdmann 2002 Design of antisense oligonucleotides stabilized by locked nucleic acids. NAR 30: 1911-1918.

    Kvaerno L, and J Wengel 1999 Investigation of restricted backbone conformation as an explanation for the exceptional thermal stabilities of duplexes involving LNA (Locked Nucleic Acid): synthesis and evaluation of abasic LNA. Chem Commun 1999, 7: 657-658.

    Latorra D, K Campbell, A Wolter, and JM Hurley 2003a Enhanced allele-specific PCR discrimination in SNP genotyping using 3’ locked nucleic acid (LNA) primers. Hum Mut 22: 79-85.

    Latorra D, K Arar, and JM Hurley 2003b Design considerations and effects of LNA in PCR primers. Mol Cell Probes 17: 253-9.

    McTigue PM, RJ Peterson, and JD Kahn 2004 Sequence-dependent thermodynamic parameters for locked nucleic acid (LNA)-DNA duplex formation. Biochemistry 43: 5388-5405.

    Yasunori Mitsuoka, Tetsuya Kodama, Ryo Ohnishi, Yoshiyuki Hari, Takeshi Imanishi and Satoshi Obika; A bridged nucleic acid, 2’,4’-BNACOC: synthesis of fully modified oligonucleotides bearing thymine, 5-methylcytosine, adenine and guanine 2’,4’-BNACOC monomers and RNA-selective nucleic-acid recognition. Nucleic Acids Research, 2009, Vol. 37, No. 4 1225–1238. Note: BNA-COC/DNA and BNA-COC/RNA duplex formation.

    Kazuyuki Miyashita, S. M. Abdur Rahman, Sayori Seki, Satoshi Obikaab and Takeshi Imanishi; N-Methyl substituted 2’,4’-BNANC: a highly nuclease-resistant nucleic acid analogue with high-affinity RNA selective hybridization. Chem. Commun., 2007, 3765–3767.

    Nielson PE, and G Haaima 1997 Peptide nucleic acid (PNA). A DNA mimic with a pseudopeptide backbone. Chem Soc Rev 26: 73-78.

    Nomenclature for polynucleotide chains including for the sugar puckering can be found at: http://www.chem.qmul.ac.uk/iupac/misc/pnuc2.html

    Satoshi Obika, Daishu Nanbu, Yoshiyuki Hari, Ken.ichiro Morio, Yasuko In, Toshimasa Ishida, and Takeshi Imanishi; Synthesis of 2'-O,4'-C-Methyleneuridine and -cytidine. Novel Bicyclic Nucleosides Having a Fixed C a ,-endo Sugar Puckering. Tetrahedron Letters, Vol. 38, No. 50, pp. 8735-8738, 1997.

    Obika S, D Nanbu, Y Hari, J-i Andoh, K-i Morio, T Doi, and T Imanishi 1998 Stability and structural features of the duplexes containing nulcoeside analogs with a fixed N-type conformation. 2’-O, 4’-C methylene ribonucleosides. Tetrahedron Lett 39: 5401-5404.

    Satoshi Obika, Mayumi Onoda, Koji Morita, Jun-ichi Andoh, Makoto Koizumi and Takeshi Imanishi; 3’-Amino-2’,4’-BNA: novel bridged nucleic acids having an N3’->P5’ phosphoramidate linkage. Chem. Commun., 2001, 1992–1993. Note: BNA/DNA; BNA/dsDNA.

    Satoshi Obika, Yoshiyuki Hari, Mitsuaki Sekiguchi, and Takeshi Imanishi; A 2',4'-Bridged Nucleic Acid Containing 2-Pyridone as a Nucleobase: Efficient Recognition of a C●G Interruption by Triplex Formation with a Pyrimidine Motif. Angew. Chem. Int. Ed. 2001, 40, No. 11, 2079-2081.

    Satoshi Obika, Mitsuaki Sekiguchi, Roongjang Somjing, and Takeshi Imanishi; Adjustment of the g Dihedral Angle of an Oligonucleotide P3’!N5’ Phosphoramidate Enhances Its Binding Affinity towards Complementary Strands. Angew. Chem. Int. Ed. 2005, 44, 1944 –1947.

    Satoshi Obika, Masaharu Tomizu, Yoshinori Negoro, Ayako Orita, Osamu Nakagawa, and Takeshi Imanishi; Double-Stranded DNA-Templated Oligonucleotide Digestion Triggered by Triplex Formation. ChemBioChem 2007, 8, 1924 – 1928. Note: Triplex triggered cleavage of oligonucleotides.

    Satoshi Obika, Hiroyasu Inohara, Yoshiyuki Hari,_ and Takeshi Imanishi; Recognition of T●A interruption by 2’,4’-BNAs bearing heteroaromatic nucleobases through parallel motif triplex formation. Bioorganic & Medicinal Chemistry 16 (2008) 2945–2954.

    Satoshi Obika, S. M. Abdur Rahman, Bingbing Song, Mayumi Onoda, Makoto Koizumi, Koji Morita, Takeshi Imanishi; Synthesis and properties of 3’-amino-2’,4’-BNA, a bridged nucleic acid with a N3’->P5’ phosphoramidate linkage. Bioorganic & Medicinal Chemistry 16 (2008) 9230–9237.

    Petersen M, and J Wengel 2003 LNA: a versatile tool for therapeutics and genomics. Trends Biotechnol 21:74-81.

    Petersen M, CB Nielsen, KE Nielsen, GA Jensen, K Bondensgaard, SJ Singh, VK Rajwanshi, AA Koshkin, BM Dahl, J Wengel, and JP Jacobsen 2000 The conformations of locked nucleic acids (LNA). J Mol Recognition 13: 44-53.

    Petersen M, JJ Sorensen, and JT Nielsen 2003 Structural basis for LNA (locked nucleic acid) triplex formation. Presented at the 5th International Congress on Molecular Structural Biology.

    S. M. Abdur Rahman, Sayori Seki, Satoshi Obika, Sunao Haitani, Kazuyuki Miyashita, and Takeshi Imanishi; Highly Stable Pyrimidine-Motif Triplex Formation at Physiological pH Values by a Bridged Nucleic Acid Analogue. Angew. Chem. Int. Ed. 2007, 46, 4306 –4309.

    S. M. Abdur Rahman, Sayori Seki, Satoshi Obika, Haruhisa Yoshikawa, Kazuyuki Miyashita, and Takeshi Imanishi; Design, Synthesis, and Properties of 2’,4’-BNANC: A Bridged Nucleic Acid Analogue.J. AM. CHEM. SOC. 2008, 130, 4886-4896.

    S. M. Abdur Rahman, Hiroyuki Sato, Naoto Tsuda, Sunao Haitani, Keisuke Narukawa, Takeshi Imanishi, Satoshi Obika; RNA interference with 2’,4’-bridged nucleic acid analogues. Bioorganic & Medicinal Chemistry 18 (2010) 3474–3480.

    Schulz RG, and SM Gryaznov 1996 Oligo-2'-fluoro-2'-deoxynucleotide N3'-->P5' phosphoramidates: synthesis and properties. NAR 24: 2966-73.

    Simeonov A, and TT Nikiforov 2002 Single nucleotide polymorphism genotyping using short, fluorescently labeled locked nucleic acid (LNA) probes and fluorescence polarization detection. NAR 30: e91.

    Singh SK, P Nielsen, AA Koshkin, and J Wengel 1998 LNA (locked nucleic acids): synthesis and high-affinity nucleic acid recognition. Chem Commun 4: 455-456.

    Tolstrup N, PS Nielsen, JG Kolberg, AM Frankel, H Vissing, and S Kauppinen 2003 OilgoDesign: optimal design of LNA (locked nucleic acid) oligonucleotide capture probes for gene expression profiling. NAR 31: 3758-3762.

    Torigoe H, Y Hari, M Sekiguchi, S Obika, and T Imanishi 2001; 2’-O, 4’-C-methylene bridged nucleic acid modification promotes pyrimidine motif triplex DNA formation at physiologic pH. J Biol Chem 276: 2354-2360. Note: TFO for therapeutics.

    Hidetaka Torigoe, Osamu Nakagawa, Takeshi Imanishi, Satoshi Obika, Kiyomi Sasaki; Chemical modification of triplex-forming oligonucleotide to promote pyrimidine motif triplex formation at physiological pH. Biochimie 94 (2012) 1032-1040. Note: pyrimidine motif triplex formation by 3’-amino-2’-O,4’-BNA.

    Ugozzoli LA, D Latorra, R Pucket, K Arar, and K Hamby 2004 Real-time genotyping with oligonucleotide probes containing locked nucleic acids. Anal Biochem 324: 143-152.

    Van Aerschot A, I Verheggen, C Hendrix,and P Herdewijn 1995 1,5-Anhydrohexitol nucleic acids, a new promising antisense construct. Angew Chem Int Ed 34: 1338–1339.

    Tsuyoshi Yamamoto, Mariko Harada-Shiba, Moeka Nakatani, Shunsuke Wada, Hidenori Yasuhara, Keisuke Narukawa, Kiyomi Sasaki, Masa-Aki Shibata, Hidetaka Torigoe, Tetsuji Yamaoka, Takeshi Imanishi and Satoshi Obika; Cholesterol-lowering Action of BNA-based Antisense Oligonucleotides Targeting PCSK9 in Atherogenic Diet-induced Hypercholesterolemic Mice Molecular Therapy–Nucleic Acids (2012) 1, e22; oi:10.1038/mtna.2012.16.
     

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    Citrullinated Peptide Synthesis

    The amino acid citrulline is not coded for by DNA directly however several proteins are known to contain citrulline as a result of a posttranslational modification. These citrulline residues are generated by a family of enzymes called peptidylarginine deiminases (PADs), which convert arginine into citrulline in a process called citrullination or deimination. It has became clear in recent years that histones, fibronectin, myelin basic protein (MBP) as well as other cellular proteins can be modified by post-translational changes during epigenetic regulation in the cell. Modifications include acetylation, methylation, phosphorylation, ubiquitination and citrullination among others. Histone modifications induce changes to the struc&amp;not;ture of chromatin and thereby affect the accessibility of the DNA strand to transcriptional enzymes, resulting in activation or repression of genes associated with modified histones. So far citrulline modifications have been connected with the autoimmune disorders multiple sclerosis and rheumatoid arthritis. Biosynthesis offers citrulline incorporation into any peptide sequence to help researchers study the effect of these modifications in vivo or in vitro.

    citrullinated peptide synthesis

    Deimination reaction of arginyl residues within peptide bonds Deimination of arginine results in neutral citrulline with the release of ammonia and the loss of one positive charge for each arginyl residue deiminated. The process is catalyzed by a calcium dependent peptidylarginine deiminase.

    Contact us for more information on Peptide Citrullin modification


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    Mycobacterium tuberculosis peptide libraries

    Design of Mtb peptide libraries for targeted research

    Tuberculosis, an ancient disease, has become an escalating global health problem in recent years. It is estimated that one-third of the world’s population presently is infected with Mycobacterium tuberculosis (Mtb) and that the bacteria causes 1.7 million deaths annually. On November 20th, 2012 the news reported that students in Grand Forks, ND, were diagnosed with tuberculosis and health officials reported three new cases of tuberculosis (TB) in Grand Forks, including a student at Valley Middle School. This brought the total number of active cases to 13. An article in the Wall Street Journal on Friday November 23, 2012 reported how the fight against TB made the bacteria stronger. Basically, the use of the standard treatment to cure TB in India and other countries with a high infection rate by giving patients a cocktail of antibiotic drugs at a cost of $9 per month without prior testing for drug-resistant strains, allowed the bacteria to evolve more resistant strains. Additional to the cost for testing in a certified lab to help guide a successful treatment, the cost to treat resistant TB strains amounts to $2,000 per month. Chances are that until an integrated approach that uses testing for drug-resistance together with a targeted treatment is established, the infection rate of TB will continue to increase worldwide.

    TB a bacterial infection caused by the bacteria Mycobacterium tuberculosis usually attack the lungs, but can also spread to other parts of the body. TB disperses into the air when a person with TB of the lungs or throat coughs, sneezes or talks. Only a medical examination will tell if a person has been exposed to the bacteria. People with weak immune systems have a higher risk for infection. Symptoms of TB in the lungs may include a bad cough that lasts 3 weeks or longer, weight loss, coughing up blood or mucus, weakness or fatigue, fever and chills, and night sweats. TB can be deadly if not treated properly and it is highly recommend that people experiencing these symptoms go right away to a doctor. Exposure to tuberculosis that includes frequent or prolonged exposure, such as sitting in a small room or confined area for a long period of time with someone who has active TB is infectious. However, people are only contagious when there is active disease in their lungs or throat that has not been treated. TB does not spread through clothes, dishes, floors or furniture. The estimate is that in India the disease kills 300,000 people out of 1.2 billion a year. Unfortunately, anyone can get TB. Individuals with weakened immune systems, including those with AIDS or those infected with HIV, have a higher risk to get infected. Active TB can usually be cured with several medicines taken for a long period of time. Latent TB can be treated to prevent the development of active TB. The standard tuberculosis treatment is to take a combination of antibiotics for half a year or more. If patients quit the therapy prematurely, the risk that drug-resistant strains emerge increases. Mycobacteria are hard to kill and it was assumed that dormant cells less susceptible to antibiotics exist even in patients with active disease. Recently a study discovered that Mycobacteria divide asymmetrically. This behavior generates a population of cells that grow at different rates, have different sizes, and differ in how susceptible they are to antibiotics. This gives the bacteria an increased chance for survival. These new findings may aid in the development of new drugs against the hard to kill Mtb cells.

    The origin of the Mtb complex, its spread and demography was studied in 2008 by Wirth et al. using a new approach that employed genetic markers based on mycobacterial interspersed repetitive units. These genetic loci comprise variable numbers and tandem repeat sequences that allowed them to be used as genotyping markers. Similar to microsatellites, they behave as selectively neutral phylogenetic markers if large numbers of loci are used. The research group used these markers to calculate a molecular clock and to model their evolution. They report that the Mtb complex consist of two independent clades. One composed of lineages from humans and a second one composed of lineages from both animal and human isolates. Furthermore, the results provide genetic evidence that the most common ancestor for this bacterial complex emerged some 40,000 years ago from its progenitor in East Africa from where it spread in parallel to human migration routes.

    The World Health Organization reports on their web site that “tuberculosis (TB) is second only to HIV/AIDS as the greatest killer worldwide due to a single infectious agent. In 2011, 8.7 million people fell ill with TB and 1.4 million died from TB. Over 95% of TB deaths occur in low- and middle-income countries. TB is among the top three causes of death for women aged 15 to 44. In 2010, there were about 10 million orphan children as a result of TB deaths among parents. TB is a leading killer of people living with HIV causing one quarter of all deaths. Multi-drug resistant TB (MDR-TB) is present in virtually all countries surveyed.” The good news is that the estimated number of people falling ill with tuberculosis each year is slowly declining. The TB death rate dropped 41% between 1990 and 2011. TB occurs in every part of the world. In 2011, the largest number of new TB cases occurred in Asia, accounting for 60% of new cases globally. Sub-Saharan Africa carried the greatest proportion of new cases per population with over 260 cases per 100 000 population in 2011. (Source: http://www.who.int/mediacentre/factsheets/fs104/en/).

    Mtb peptide libraries can be used to help developing new vaccines against TB

    There is an urgent need to design new antituberculosis vaccines. The anti-TB vaccine presently available has a high variability and is ineffective in adults. The goal for immunologist is to design a vaccine that confers complete protection against the disease. Presently, research is focused on finding new Mtb-specific antigens that can be used to replace or improve the old vaccine. Early innate and adaptive responses to Mtb are critical for the successful containment of the infection. However, these events are poorly understood, in part because low-dose aerogenic inoculation does not trigger a robust early inflammatory response. Complicating this is the fact that very early T-cell responses are difficult to study. There remain many open questions such as if Mtb delays the onset of early immunity, either by accident or design, and if true memory T lymphocytes can be elicited by vaccination. New research tools are now available to help improve our understanding of TB immunity. Studying the host genetic factors such as human leucocyte antigens (HLA) and non-HLA genes/gene products that are associated with the susceptibility to TB has the potential to determine genetic markers that help in understanding predisposition factors that allow for the development of the disease. Establishing a clear picture of the immune response network to this pathogen is essential for the design of effective vaccines. Identification of high-activity binding peptides that are able to inhibit bacterial invasion is a step toward this goal. Ocampo et al. in 2012 report the identification of Mtb Rv3166c protein high-activity binding peptides (HABPs) that were able to inhibit bacterial invasion of U937 (monocyte-derived macrophages) and A549 (type II alveolar epithelial cells) cell lines. The researchers used PCR to confirm the presence and transcription of the rv3166c gene in the Mtb species complex. Western blotting and immunoelectron microscopy was used to evaluate and confirm Rv3166c expression which was found to be present mainly on the cell surface. The research group synthesized sixteen 21mer peptides that covered the entire length of the protein and contained a tyrosine on the C-terminal end. The tyrosine allowed the radiolabeling of the peptides after synthesis using I125 to be used in a binding assay. The peptides were tested for their ability to bind to U937 and A549 cells. Two U937 HABPs were identified and three for A549, one of them being shared by both cell lines. Four peptides were identified to inhibited Mtb entry by 15.07–94.06%. These results led the researchers to conclude that Rv3166c HABPs can be used as candidates for further studies contributing towards the search for a multiepitope, chemically synthesized, subunit-based antituberculosis vaccine. The next table lists the peptides used for the study.

    The graph shows the Rv3166c peptide binding activity profile. The amino acid sequences and specific U937 and A549 cell binding activities for Rv3166c peptides are listed. Tyr was added at the C-terminal end of those peptides lacking it to facilitate I125 radiolabeling. The black bars on the right-hand side represent each peptide’s binding activity determined as the specific binding/total peptide added ratio. The dotted line separates peptides having ≥1% binding activity (Ocampo et al.).

    The key to generating good experimental results is the design of the experimental approach on how to use scientific tools such as peptide libraries. Below is the description of the design of peptide libraries or pools using a few selected examples.

    Example of peptide library design using the sequence of the hypothetical protein Rv3166c [Mycobacterium tuberculosis H37Rv] NP_217682.1:

    1
    MPGTKPGSDK PTGRVVVVIV LLMLAGAALR GHLPADDGAP LAAAGGSRAA LMFIVAALAA
    61
    TLALIALAII TRLRHPLPVA PSAGELSAML GGAAGRPNWR VLLLGLGTIL AWLLIAILLA
    121
    RLFVPDDVGP AAPIPDSTAT PDASSTTPSR PQPPQDNNDD VLGILFASTI GLFLMVVAGS
    181
    LITSRRQRKS APARISGDRI ESPAPSARSE SLARAAEIGL AEMADLRREP REAIIACYVA
    241
    MERELSHVPG VAPQDFDTPT EVLARAVEHR ALHGASAAAL VSLFAEARFS PHVMNEEHRE
    301
    VAMRLLRLVL DELSTRTAI        

    1. Non over lapping peptide libraries:

    This example shows the design of a non overlapping library. First, select the sequences of peptides to be synthesized. In this case 20mer peptides are selected highlighted in alternating grey shadowing.

    1
    MPGTKPGSDK PTGRVVVVIV LLMLAGAALR GHLPADDGAP LAAAGGSRAA LMFIVAALAA
    61
    TLALIALAII TRLRHPLPVA PSAGELSAML GGAAGRPNWR VLLLGLGTIL AWLLIAILLA
    121
    RLFVPDDVGP AAPIPDSTAT PDASSTTPSR PQPPQDNNDD VLGILFASTI GLFLMVVAGS
    181
    LITSRRQRKS APARISGDRI ESPAPSARSE SLARAAEIGL AEMADLRREP REAIIACYVA
    241
    MERELSHVPG VAPQDFDTPT EVLARAVEHR ALHGASAAAL VSLFAEARFS PHVMNEEHRE
    301
    VAMRLLRLVL DELSTRTAI        

    The library for this selected protein consists of 16 peptides. The selected peptides can be modified either on the N- or C-terminal end with tyrosine to allow for the use of I125 as the radiolabel, cysteine, to allow for conjugation to another compound or to beads, biotin, to allow for binding to avidin or streptavidin coated beads, or any other desired label such as a fluorophore or stable isotope tag, to allow for the coding of the peptides. The use for the downstream analysis tools will determine what type of label needs to be selected.

    2. Over-lapping peptide libraries:

    The next example shows the design of a library that uses 20mer peptides and an overlap of 11 amino acid residues. This type of peptide library can be designed by using the whole sequence of the original protein or protein domain to generate many equal-length overlapping peptide fragments. Typical applications are continuous epitope mapping and T-cell epitope determination. The “Peptide Library Tools” that can be found on Biosynthesis Inc.’s web site was used for the design. The following link shows the location of the tool: http://www.biosyn.com/PeptideDesignLibrary/PeptideDesignLibrary.aspx.

    The resulting list of selected peptides is shown in the following table.
     

    #

    Sequence

    Position

    Mw

    Hydrophilicity Ratio

    1

    MPGTKPGSDKPTGRVVVVIV

    1-20

    2037.44

    25

    2

    KPTGRVVVVIVLLMLAGAAL

    10-29

    2020.58

    10

    3

    IVLLMLAGAALRGHLPADDG

    19-38

    2003.39

    15

    4

    ALRGHLPADDGAPLAAAGGS

    28-47

    1816.99

    20

    5

    DGAPLAAAGGSRAALMFIVA

    37-56

    1859.18

    15

    6

    GSRAALMFIVAALAATLALI

    46-65

    1973.46

    10

    7

    VAALAATLALIALAIITRLR

    55-74

    2034.57

    10

    8

    LIALAIITRLRHPLPVAPSA

    64-83

    2122.64

    15

    9

    LRHPLPVAPSAGELSAMLGG

    73-92

    1973.33

    20

    10

    SAGELSAMLGGAAGRPNWRV

    82-101

    2000.26

    30

    11

    GGAAGRPNWRVLLLGLGTIL

    91-110

    2034.42

    15

    12

    RVLLLGLGTILAWLLIAILL

    100-119

    2174.82

    5

    13

    ILAWLLIAILLARLFVPDDV

    109-128

    2264.82

    15

    14

    LLARLFVPDDVGPAAPIPDS

    118-137

    2063.39

    25

    15

    DVGPAAPIPDSTATPDASST

    127-146

    1869.98

    30

    16

    DSTATPDASSTTPSRPQPPQ

    136-155

    2041.15

    45

    17

    STTPSRPQPPQDNNDDVLGI

    145-164

    2151.28

    50

    18

    PQDNNDDVLGILFASTIGLF

    154-173 

    2149.38

    35

    19

    GILFASTIGLFLMVVAGSLI

     163-182 

    2022.52

    10

    20

    LFLMVVAGSLITSRRQRKSA

     172-191 

    2233.71

    40

    21

    LITSRRQRKSAPARISGDRI

     181-200 

    2281.66

    55

    22

    SAPARISGDRIESPAPSARS

     190-209 

    2025.23

    50

    23

    RIESPAPSARSESLARAAEI

     199-218 

    2111.37

    50

    24

    RSESLARAAEIGLAEMADLR 

    208-227 

    2159.47

    45

    25

    EIGLAEMADLRREPREAIIA 

    217-236 

    2253.63

    40

    26

    LRREPREAIIACYVAMEREL

    226-245 

    2418.89

    40

    27

    IACYVAMERELSHVPGVAPQ 

    235-254 

    2170.54

    25

    28

    ELSHVPGVAPQDFDTPTEVL 

    244-263

    2151.37

    30

    29

    PQDFDTPTEVLARAVEHRAL

    253-272 

    2265.53

    35

    30

    VLARAVEHRALHGASAAALV  

    262-281

    2012.34

    20

    31

    ALHGASAAALVSLFAEARFS 

    271-290 

    1989.27

    25

    32

    LVSLFAEARFSPHVMNEEHR 

    280-299 

    2369.7

    40

    33

    FSPHVMNEEHREVAMRLLRL 

    289-308 

    2464.91

    40

    34

    HREVAMRLLRLVLDELSTRT 

    298-317 

    2408.87

    40

    35

    RLVLDELSTRTAI   

    307-319 

    1486.74

    38.46


    Many more permutations of peptide library designs are possible. The resulting libraries can be used to screen highly active compounds such as antigenic peptides, receptor ligands, antimicrobial compounds, protein binding interfaces, and enzyme inhibitors among others.

    Major applications are the use of peptide libraries for epitope mapping studies, vaccine research, high-throughput protein interaction analysis, customized peptide microarray production and kinase assays. Epitope mapping requires the design of overlapping peptide libraries, which can be customized by adjusting the fragment length and offset number for the optimum balance between low costs and high data value.

    Next, the different library types are explained.

    Alanine Peptide Scanning Library: The design of a peptide library in which alanine (Ala, A) is systematically substituted into each of the amino acids which can be used to identify epitope activity. This is sometimes also called an alanine scanning library.

    Positional Peptide Library: A selected position in a peptide sequence is systematically replaced with a different amino acid to study the effect of the substituted amino acid at a certain position.

    Truncation Peptide Library: A truncation library can be used to predict the minimum amino acid sequence length required for optimum epitope activity.

    Random Peptide Library: To design this library selected positions within the original peptide sequence are randomly substituting other natural amino acids in a shot-gun type approach help find potential alternative sequences for enhanced peptide activity.

    Scramble Peptide Library: A scrambled library is designed by carrying out permutation on the original peptide's sequence. It has the potential to give all possible alternatives and offers and represents the highest degree of variability per peptide library.

    T-cell Truncated Peptide Libraries: This library allows the testing of all possible T-cell epitopes across a protein of interest.

    Examples of Mtb peptide libraries and there use found in the literature

    Kovjazin in 2010 reported the use of bioinformatic methods to find Mtb peptides with high epitope densities that can be used for vaccine design. Signal peptides and trans-membrane domains were found to have exceptionally high epitope densities. The researchers explain that the major histocompatibility complex (MHC)-binding of these domains relies on their hydrophobic nature and their specific sequence. The research group computed the epitope densities for signal peptides and experimentally confirmed their immunogenicity using a panel of nine synthetic peptides in vitro. The scientists reason that the high epitope density may be the result of an arbitrary overlap between the typical patterns of signal peptides and frequent MHC binding motifs. And that the high epitope density in such domains could be explained on the basis of an evolutionary advantage caused by preferential presentation of hydrophobic sequences especially for the signal peptides. It is known that hostpathogen coevolutionary pressures can change the relative frequency of the different HLA alleles in a population and it is thus possible that HLA alleles preferentially presenting signal peptide epitopes were selected. Highly conserved signal peptide motifs are almost similar in Eukaryotes and Prokaryotes. The authors reason that these results may indicate that pathogens use secreted proteins to manipulate their hosts, and that the host has evolved a mechanisms to present bacterial peptides on MHC-I molecules.

    Joosten et al. in 2010 describe responses of human T-cells to Mtb-derived peptides containing predicted HLA-E binding motifs. A total of 69 peptides were used for the study. The CD8+ T-cell is one player of the immune response against Mtb. CD8+ T-cells recognize infected cells through Mtb derived peptides that are presented on HLA class I molecules. The nonclassical HLA molecule HLA-E was studied as presenter of Mtb antigens. The Mtb genome contains multiple sequences that can be presented by human HLA-E. These peptides were recognized by CD8+ T-cells from healthy individuals that were sensitized to Mtb, resulting in CD8+ T-cell proliferation. The investigated T-cells were able to lyse mycobacterium infected cells in a HLA-E restricted manner. Additionally, these T-cells also inhibited proliferation of other T-cells in their vicinity, which is a property of regulatory T-cells.

    Dye and Williams report in 2010 that M. tuberculosis is a family of strains and lineages and that the TB epidemiology is changing in the world today. Recent investigations have revealed hypervariable regions in the genome, the African origins and the spread around the world of the pathogen, and which genes code for drug resistance. The data demonstrated that different strains spread more or less quickly through populations and are more or less capable to cause active TB. This indicates that the magnitude of the variations and its epidemiological consequences are mostly unquantified. The analysis of different strains to determine their genetics and phenotypic differences may be needed to allow for a better control of the disease in the future.
     

    Russel et al in 2010 describe the life cycle of M. Tuberculosis in the journal Science. They state that “There is no effective vaccine against infection, and current drug therapies are fraught with problems, predominantly because of the protracted nature of the treatment and the increasing occurrence of drug resistance.” The life cycle of M. tuberculosis (see figure) begins when Mtb bacilli, present in exhaled droplets or nuclei, are inhaled and engulfed and digested by resident alveolar macrophages initiate the infection. The result is a pro-inflammatory response which triggers the infected cells to invade the epithelial tissue. Monocytes are recruited from the circulation and extensive neovascularization occurs in the infection site. Neovascularization refers to the formation of functional microvascular networks with red blood cell perfusion. The macrophages in the granulomas differentiate to form epithelioid cells, multinucleate giant cells, and foam cells filled with lipid droplets. The granuloma, a tiny collection of

    immune cells known as macrophages, can become further stratified by the formation of a fibrous extra layer of extracellular matrix material that is laid down outside the macrophage layer. Lymphocytes appear to be restricted primarily to this peripheral area. Many of the granulomas persist in this balanced state, but progression toward disease is characterized by the loss of vascularization, increased necrosis, and the accumulation of caseum in the granuloma center. Ultimately, infectious bacilli are released into the airways when the granuloma cavitates and collapses into the lungs. (Taken from Russel at al. 2010). According to the paper the research field is lacking some of the most basic tools to evaluate or assessing the beneficial effect of new drugs. Furthermore, no clear biomarkers to assess the disease status are presently available.

     

    Chun et al. in 2001 report the use of N-formylated Mtb peptides to test their ability to bind M3 using an immunofluorescence-based peptide-binding assay. M3 is an MHC class Ib molecule that preferentially presents N-formulated peptides to CD8+ T cells. Bacteria initiate protein synthesis with N-formylated methionine which makes M3 especially suitable for presenting this types of peptide epitopes. Therefore, the research group scanned the full sequence of the Mtb genome for N-terminal peptides that shared common features with other M3-binding peptides using bioinformatic tools. Synthetic peptides corresponding to the selected sequences were tested for their ability to bind M3. Furthermore, the researchers report that four of these peptides were able to elicit cytotoxic T lymphocytes (CTLs) from mice immunized with peptide-coated splenocytes. They concluded that their data suggest that M3-restricted T cells may participate in the immune response to Mtb.


    References
     

    Taehoon Chun, Natalya V. Serbina, Dawn Nolt, Bin Wang, Nancy M. Chiu, JoAnne L. Flynn, and Chyung-Ru Wang; Induction of M3-restricted Cytotoxic T Lymphocyte Responses by N-formylated Peptides Derived from Mycobacterium tuberculosis. J. Exp. Med. Volume 193, Number 10, May 21, 2001 1213–1220.

    Christopher Dye and Brian G. Williams; The Population Dynamics and Control of Tuberculosis. Science 328, 856 (2010).
    Simone A. Joosten, Krista E. van Meijgaarden, Pascale C. van Weeren, Fatima Kazi, Annemieke Geluk, Nigel D. L. Savage; Mycobacterium tuberculosis Peptides Presented by HLA-E Molecules Are Targets for Human CD8+ T-Cells with Cytotoxic as well as Regulatory Activity. PLoS Pathogens | www.plospathogens.org 1 February 2010 | Volume 6 | Issue 2 | e1000782.
     
    Riva Kovjazina, Ilan Volovitzc, Yair Daona, Tal ViderShalitb, Roy Azranb, Lea Tsabanb, Lior Carmona, Yoram Louzoun; Signal peptides and transmembrane regions are broadly immunogenic and have high CD8+ T cell epitope densities: Implications for vaccine development. Molecular Immunology 48 (2011) 1009–1018.
     
    Frieder M, Lewinsohn DM. T-cell epitope mapping in Mycobacterium tuberculosis using pepmixes created by micro-scale SPOT- synthesis. Methods Mol Biol. 2009;524:369-82.
    Marisol Ocampo, Daniel Aristiza´bal-Ramı´rez, Diana M.Rodrı´guez, Marina Mun˜oz, Hernando Curtidor, Magnolia Vanegas, Manuel A.Patarroyo, and Manuel E.Patarroyo; The role of Mycobacterium tuberculosis Rv3166c protein-derived high-activity binding peptides in inhibiting invasion of human cell lines. Protein Engineering, Design & Selection vol. 25 no. 5 pp. 235–242, 2012.
     
    David G. Russell, Clifton E. Barry 3rd, JoAnne L. Flynn; Tuberculosis: What We Don't Know Can, and Does, Hurt Us. Science 328, 852 (2010).
    Winslow GM, Cooper A, Reiley W, Chatterjee M, Woodland DL; Early T-cell responses in tuberculosis immunity. Immunol Rev. 2008 Oct; 225:284-99.

    Wirth T, Hildebrand F, Allix-Béguec C, Wölbeling F, Kubica T, et al. (2008) Origin, Spread and Demography of the Mycobacterium tuberculosis Complex. PLoS Pathog 4(9): e1000160. doi:10.1371/journal.ppat.1000160
     

     


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    A brief history of Bridged Nucleic Acids (BNAs)

    A quest for better oligonucleotide mimics.

    The quest for oligonucleotide mimics with improved characteristics and stabilities useful for molecular diagnostics and therapeutics that also show minimal side effects has led to the design and synthesis of novel bridged nucleic acid monomers and oligonucleotides. These synthetic oligonucleotide mimics may be used as tools for gene validation, as antisense (targeting mRNA) and antigene (targeting DNA) agents, for selective regulation of gene expression and as a potentially new class of drugs for the treatment of diseases such as cancer, inflammation, viral diseases and other pathological diseases. The 3D structures for A-RNA and B-DNA were used as a template for the design of the BNA monomers. The goal for the design is to find derivatives that posses high binding affinities with complementary RNA and/or DNA strands.


    RNA contains ribose rather than 2’-deoxyribose in its backbone. The ribose has a hydroxyl group at the 2’-position. Furthermore, RNA contains the nucleic acid uracil in place of thymine and is usually found as a single polynucleotide chain. While RNA is typically single stranded, RNA chains can frequently fold back on themselves to form base-paired segments between short stretches of complementary sequences. The presence of 2’-hydroxyls in the RNA backbone favors a structure that resembles the A-form structure of DNA. The flexible five-membered

    furanose ring in nucleotides exists in equilibrium of two preferred conformations of the N- (C3’-endo, A-form) and the S-type (C2’-endo, B-form) as depicted in the insert. A closer look at the two forms is shown below. An increased conformational inflexibility of the sugar moiety in nucleosides (oligonucleotides) should result in a gain of high binding affinity with complementary single-stranded RNA and/or double-stranded DNA.

    Synthetic oligonucleotides are now important, established tools for life scientists and have many applications in molecular biology and genetic diagnostics, and are poised to become important tools in the emerging field of molecular medicine as well. While unmodified oligodeoxynucleotides can form DNA:DNA and DNA:RNA duplexes they are sometimes unstable and labile to nucleases. Therefore a variety of nucleic acid analogs have been developed to enhance high-affinity recognition of DNA and RNA targets, enhancing duplex stability and assist with cellular uptake. 

     

    Bridged nucleic acids (BNAs) are molecules that contain a five-membered or six-membered bridged structure with a “fixed” C3’-endo sugar puckering (Saenger 1984). The bridge is synthetically incorporated at the 2’, 4’-position of the ribose to afford a 2’, 4’-BNA monomer. The monomers can be incorporated into oligonucleotide polymeric structures using standard phosphoamidite chemistry. BNAs are structurally rigid oligo-nucleotides with increased binding affinities and stability. 

    The incorporation of BNAs into oligonucleotides allows the production of modified synthetic oligonucleotides with:

    1. Equal or higher binding affinity against an DNA or RNA complement with excellent single-mismatch discriminating power,

    2. Better RNA selective binding,

    3. Stronger and more sequence selective triplex-forming characters, and

    4. Pronounced higher nuclease resistance, even higher than Sp-phosphorthioate analogues,

    5. Good aqueous solubility of the resulting oligonucleotides when compared to regular DNA or RNA oligonucleotides

    BNAs can be synthesized using standard phosphoramidite chemistry.

    The first synthesis of bridged 2’-O, 4’-C-methyleneuridine and –cytidine monomers were described by Obika et al. in 1997 (in Imanishi’s group). The same group showed in 1998 that these monomers allowed the formation of stable oligonucleotide duplexes in both DNA and RNA based synthetic 12 meric oligonucleotides. Chemical structures for nucleosides and a bridged nucleoside are shown below.

    Koshkin et al. in 1998 demonstrated that these monomers can be used to synthesize oligonucleotides that can form stable complexes with DNA and RNA oligonucleotides. Furthermore, the group gave these monomers a new name and called them “Locked Nucleic Acids” (LNAs). The synthesis of these bridged nucleic acids could be achieved by standard phosphoramidite chemistry.

    Jesper Wengel in 1999 describes the synthesis of 3’-C- and 4’-C-branched oligonucleotides and the development of locked nucleic acids as well as their use as DNA/RNA mimics. 

    Christensen et al. in 2001 used stopped-flow kinetic measurements to study the thermodynamics of LNA oligonucleotide based complexes.  

    Obika at al. in 2001 report that 2'-O, 4'-C-methylene bridged nucleic acids (2',4'-BNAs = LNA) have unprecedented binding affinities towards their complementary RNA. The researchers showed that 2',4'-BNA oligonucleotides can be used as antisense molecules and demonstrated their potent inhibitory effect on gene expression of Intercellular Adhesion Molecule-1 (ICAM-1) in living cells. Furthermore, the contribution of RNase H to this antisense effect and adequate stability of 2',4'-BNA oligonucleotides to enzymatic degradation were also demonstrated. These results showed that BNAs can be used to find natural RNA sequences and target them for destruction.


    Torigoe et al. also in 2001 report that 2'-O, 4'-C-methylene bridged nucleic acids (LNAs) can be used to synthesize modified oligonucleotides that can form triplexes with DNA at physiological pH. LNAs are the best studied and characterized bridged nucleic acids so far.

     

    Also in 2001 Obika et al. introduced a 3’-amino-2’,4’-BNA monomer and a 2’,4’-BNA that contained a 2-pyridone group as the base that showed duplex and triplex forming abilities when used in oligonucleotides. 

    Another bridged nucleic acid monomer was synthesized and introduced in 2001 by Morita et al. called 2’-O, 4’-C-ethylene-bridged nucleic acid (ENA). The 2'-O,4'-C-ethylene linkage of these nucleosides restricts the sugar puckering to the N-conformation. The ethylene-bridged nucleic acids showed a high binding affinity for the complementary RNA strand (ΔTm = +5°C per modification) and were approximately 400 and 80 times more nuclease-resistant than natural DNA and BNA/LNA, respectively. These results indicate that ENA have better antisense activities than BNA/LNA.

     

    Hari et al. in 2003 developed a novel nucleoside analogue that allowed for the effective recognition of CG interruption in a homopurine–homopyrimidine tract of double-stranded DNA (dsDNA). The scientists succeeded in the synthesis of a triplex-forming oligonucleotide (TFO) containing the novel 2’,4’-BNA (QB) bearing 1-isoquinolone as a nucleobase. The triplex-forming ability and sequence-selectivity of the TFO (TFO-QB) were examined. Melting temperature (Tm) measurements found that the TFO-QB formed a stable triplex DNA in a highly sequence-selective manner under near physiological conditions.

     

    Tolstop et al. in 2003 published a paper that described a software tool called “OligoDesign” that allowed for the ‘in-silico” design of LNA based oligonucleotides. The software provides optimal design of LNA (locked nucleic acid) substituted oligonucleotides for functional genomics applications. The OligoDesign software features recognition and filtering of the target sequence by genome-wide BLAST analysis in order to minimize cross-hybridization with non-target sequences. Routines for prediction of melting temperature, self-annealing and secondary structure for LNA substituted oligonucleotides, as well as secondary structure prediction of the target nucleotide sequence are included. Individual scores for all these properties are calculated for each possible LNA oligonucleotide in the query gene and the OligoDesign program ranks the LNA capture probes according to a combined fuzzy logic score and finally returns the top scoring probes to the user in the output. The OligoDesign program is freely accessible at http://lnatools.com/

     

    The bioinformatics tools was designed to optimize the design of modified oligonucleotides used for the following applications:

    1. microarray probes,
    2. probes for in situ hybridization,
    3. oligonucleotides for antisense inhibition,
    4. FISH probes,
    5. SNP detection as well as others

     

    Antisense oligonucleotides that contain LNAs show improved silencing potency but cause significant hepatoxicity in animals. This was noticed in 2006 by Swayze at el. when designing antisense oligonucleotides for the silencing of TRADD and ApoB genes in cell cultures. These results indicated that LNAs may need to be used with caution for antisense purposes. These characteristics led to design newer generations of BNAs.

    Miyashita et al. in 2007 (in Imanishi’s group) report the design and synthesis of a new type of BNA, a N-methyl substituted 2’,4’-BNANC. This is a highly nuclease-resistant nucleic acid analogue with high-affinity RNA selective hybridization. The monomer was designed to fine tune the BNA structure.

    The research group synthesized a novel bridged nucleic acid 2’,4’-BNANC[N–Me] and showed that it has high-affinity hybridization similar to that of 2’,4’-BNA (LNA) against an RNA complement. Furthermore, the scientists report that, the nucleic acid analogue displayed RNA selectivity superior to that of 2’,4’-BNA (LNA) and other structural analogues of 2’,4’-BNA (LNA). Nuclease resistance of this nucleic acid analogue is abundantly higher than that of 2’,4’-BNA (LNA) and also slightly higher than that of a phosphorthioate. The hydrophobic methyl substituent on the backbone might present an additional advantage resulting in cellular uptake of the oligonucleotides. All of these reported characteristics of the BNA are essential for antisense applications. In the same year Rahman et al. report that 2’,4’-BNANC form highly stable pyrimidine-motif DNA triplexes at physiological pH. These triplexes are involved in the regulation of gene expression, site-specific cleavage of DNA, gene mapping and isolation, maintenance of folded chromosome confromations, and gene-targeted mutagenesis. In a pyrimidine-motif triplex DNA the triplex forming oligonucleotide binds with the homopurine tract of the target duplex DNA in a sequence specific manner through Hoogsteen hydrogen bonds to form T●A:T and C+●G:C triads. In the same year Obika et al report that 5’-amino-BNAs can be used to digest oligonucleotides triggered by triplex formation.

     

    In 2008 Imanishi’s group (Rahman et al. 2008) introduced three new bridged nucleic acid analogues called 2’,4’-BNANC[NH], 2’,4’-BNANC[NMe], and 2’,4’-BNANC[NBn]. Structures of these analogs are shown below. The new analogs were designed by taking the length of the bridged moiety into account. A six-membered bridged structure with a unique structural feature (N-O bond) in the sugar moiety was designed to have a nitrogen atom. This atom can act as a conjugation site and improve the formation of duplexes and triplexes by lowering the repulsion between the negatively charged backbone phosphates. Furthermore, the nitrogen atom on the bridge can be functionalized by hydrophobic and hydrophilic groups, by adding groups that introduce steric bulk or any desired functional moiety. These modifications allow to control affinity towards complementary strands, regulate resistance against nuclease degradation and the synthesis of functional molecules designed for specific applications in genomics. The properties of these analogs were investigated and compared to those of previous 2’,4’-BNA (LNA) modified oligonucleotides.

    Compared to 2’,4’-BNA (LNA)-modified oligonucleotides, 2’,4’-BNANC congeners were found to possess:

    1. Equal or higher binding affinity against an RNA complement with excellent single-mismatch discriminating power,
    2. Much better RNA selective binding,
    3. Stronger and more sequence selective triplex-forming characters, and
    4. Immensely higher nuclease resistance, even higher than the Sp-phosphorthioate analogue.

    The researchers state that “2’,4’-BNANC-modified oligonucleotides with these excellent profiles show great promise for applications in antisense and antigene technologies.”

    More recently Yamamoto et al. in 2012 demonstrated successfully that BNA-based antisense therapeutics inhibited hepatic PCSK9 expression, resulting in a strong reduction of the serum LDL-C levels of mice. The findings support the hypothesis that PCSK9 is a potential therapeutic target for hypercholesterolemia. This appears to be the first time that researchers were able to show that BNA-based antisense oligonucleotides (AONs) induced cholesterol-lowering action in hypercholesterolemic mice. A moderate increase of aspartate aminotransferase, ALT, and blood urea nitrogen levels was observed whereas the histopathological analysis revealed no severe hepatic toxicities. The same group, also in 2012, report that the 2’,4’-BNANC[NMe] analog when used in antisense oligonucleotides showed significantly stronger inhibitory activities which is more pronounced in shorter (13- to 16mer) oligonucleotides. Their data led the researchers to conclude that the 2’,4’-BNANC[NMe] analog may be a better alternative to conventional LNAs.

    Action mechanism of antisense oligonucleotides

    The proposed action mechanism for antisense oligonucleotides may involve translation arrest, mRNA degradation mediated by RNase H and splicing arrest. This is illustrated in the following figure.

     

    References

    Ulla CHRISTENSEN, Nana JACOBSEN., Vivek K. RAJWANSHI, Jesper WENGEL and Troels KOCH. Stopped-flow kinetics of locked nucleic acid (LNA)–oligonucleotide duplex formation : studies of LNA–DNA and DNA–DNA interactions Biochem. J. (2001) 354, 481-484.

    Yoshiyuki Hari, Satoshi Obika, Mitsuaki Sekiguchi and Takeshi Imanishi; Selective recognition of CG interruption by 2’,4’-BNA having 1-isoquinolone as a nucleobase in a pyrimidine motif triplex formationqTetrahedron 59 (2003) 5123–5128.

    Makoto KOIZUMI; 2’-O,4’-C-Ethylene-Bridged Nucleic Acids (ENATM) as Next-Generation Antisense and Antigene Agents. Biol. Pharm. Bull. 27(4) 453-456 (2004).

    Koizumi M.; ENA oligonucleotides as therapeutics. Curr Opin Mol Ther. 2006 Apr;8(2):144-9.  

    Koshkin AA, SK Singh, P Nielsen, VK Rajwanshi, R Kumar, M Meldgaard, CE Olsen, and J Wengel 1998 LNA (Locked Nucelic Acid): Synthesis of the adenine, cytosine, guanine, 5-methylcytosine, thymine and uracil bicyclonucleoside monomers, oligomerisation, and unprecedented nucleic acid recognition. Tetrahedron 54: 3607-3630.

    Koshkin, A.A., Nielsen, P., Meldgaard, M., Rajwanshi, V. K., Singh S. K. and Wengel, J. (1998). LNA (Locked Nucleic Acid): An RNA Mimic Forming Exceedingly Stable LNA:LNA Duplexes. J. Am. Chem. Soc. 120, 13252 – 13253.

    Kazuyuki Miyashita, S. M. Abdur Rahman, Sayori Seki, Satoshi Obikaab and Takeshi Imanishi;   N-Methyl substituted 2’,4’-BNANC: a highly nuclease-resistant nucleic acid analogue with high-affinity RNA selective hybridization.  Chem. Commun., 2007, 3765–3767.

    Koji Morita, Chikako Hasegawa, Masakatsu Kaneko, Shinya Tsutsum P, Junko Sone, Tomio Ishikawa, Takeshi Imanishi and Makoto Koizumi; 2'-O,4'-C-Ethylene-bridged nucleic acids (ENA) with nuclease resistance and high affinity for RNA.  2001. Nucleic Acids Research Supplement No. 1 241-242.

    Nomenclature for polynucleotide chains including for the sugar puckering can be found at:  http://www.chem.qmul.ac.uk/iupac/misc/pnuc2.html

    Satoshi Obika, Daishu Nanbu, Yoshiyuki Hari, Ken.ichiro Morio, Yasuko In, Toshimasa Ishida, and Takeshi Imanishi; Synthesis of 2'-O,4'-C-Methyleneuridine and -cytidine. Novel Bicyclic Nucleosides Having a Fixed C3 ,-endo Sugar Puckering. Tetrahedron Letters, Vol. 38, No. 50, pp. 8735-8738, 1997.

    Obika S, D Nanbu, Y Hari, J-i Andoh, K-i Morio, T Doi, and T Imanishi 1998. Stability and structural features of the duplexes containing nulcoeside analogs with a fixed N-type conformation. 2’-O, 4’-C methylene ribonucleosides. Tetrahedron Lett 39: 5401-5404.

    Satoshi Obika,Mayumi Onoda,Koji Morita,Jun-ichi Andoh,Makoto Koizumiand Takeshi Imanishi; 3’-Amino-2’,4’-BNA: novel bridged nucleic acids having an N3’->P5’ phosphoramidate linkage. Chem. Commun., 2001, 1992–1993. Note: BNA/DNA; BNA/dsDNA.

    Satoshi Obika, Yoshiyuki Hari, Mitsuaki Sekiguchi, and Takeshi Imanishi; A 2',4'-Bridged Nucleic Acid Containing 2-Pyridone as a Nucleobase: Efficient Recognition of a C●G Interruption by Triplex Formation with a Pyrimidine Motif.  Angew. Chem. Int. Ed. 2001, 40, No. 11, 2079-2081.

    Obika S, Hemamayi R, Masuda T, Sugimoto T, Nakagawa S, Mayumi T, Imanishi T.;  Inhibition of ICAM-1 gene expression by antisense 2',4'-BNA oligonucleotides. Nucleic Acids Res Suppl. 2001;(1):145-6.

    Satoshi Obika, Masaharu Tomizu, Yoshinori Negoro, Ayako Orita, Osamu Nakagawa, and Takeshi Imanishi;  Double-Stranded DNA-Templated Oligonucleotide Digestion Triggered by Triplex Formation. ChemBioChem 2007, 8, 1924 – 1928. Note: Triplex triggered cleavage of oligonucleotides.

    S. M. Abdur Rahman, Sayori Seki, Satoshi Obika, Sunao Haitani, Kazuyuki Miyashita, and Takeshi Imanishi; Highly Stable Pyrimidine-Motif Triplex Formation at Physiological pH Values by a Bridged Nucleic Acid Analogue. Angew. Chem. Int. Ed. 2007, 46, 4306 –4309.

    Saenger, W.; Principles of Nucleic Acid Structure, Springer-Verlag, new York, 1984. 

    Eric E. Swayze,* Andrew M. Siwkowski, Edward V. Wancewicz, Michael T. Migawa, Tadeusz K. Wyrzykiewicz, Gene Hung, Brett P. Monia, and C. Frank Bennett;  Antisense oligonucleotides containing locked nucleic acid improve potency but cause significant hepatotoxicity in animals Nucleic Acids Res. 2007 January; 35(2): 687–700.

    Torigoe H, Y Hari, M Sekiguchi, S Obika, and T Imanishi 2001; 2’-O, 4’-C-methylene bridged nucleic acid modification promotes pyrimidine motif triplex DNA formation at physiologic pH. J Biol Chem 276: 2354-2360. Note: TFO for therapeutics.

    Tsuyoshi Yamamoto, Mariko Harada-Shiba, Moeka Nakatani, Shunsuke Wada, Hidenori Yasuhara, Keisuke Narukawa, Kiyomi Sasaki, Masa-Aki Shibata, Hidetaka Torigoe, Tetsuji Yamaoka, Takeshi Imanishi and Satoshi Obika;  Cholesterol-lowering Action of BNA-based Antisense Oligonucleotides Targeting PCSK9 in Atherogenic Diet-induced Hypercholesterolemic Mice Molecular Therapy–Nucleic Acids (2012) 1, e22; oi:10.1038/mtna.2012.16.

    Tsuyoshi Yamamoto, Hidenori Yasuhara, FumitoWada, Mariko Harada-Shiba, Takeshi Imanishi, and Satoshi Obika; Superior Silencing by 2’,4’-BNANC-Based Short Antisense Oligonucleotides Compared to 2’,4’-BNA/LNA-Based Apolipoprotein B Antisense Inhibitors. Hindawi Publishing Corporation, Journal of Nucleic Acids Volume 2012, Article ID 707323, 7 pages doi:10.1155/2012/707323.

    Yong You, Bernardo G. Moreira, Mark A. Behlke, and Richard Owczarzy; Design of LNA probes that improve mismatch discrimination. Nucleic Acids Research, 2006, Vol. 34, No. 8 e60.


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    We report a method for the site-specific deposition of gold nanoparticles, as programmed by DNA sequences immobilized on the surface of silicon oxide-coated silicon wafers. After optimization of surface chemistries, selectivities of between 8 : 1 and 118 : 1 were achieved for the DNA-based sorting of populations of gold nanoparticle of 15 nm and 60 nm diameter from a common suspension via oligonucleotide duplex formation.

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    Design of BNANC [NMe] modified oligonucleotides

    The placing of BNA NC [NMe] monomers in oligonucleotides, both, for DNA and RNA, is shown based on examples from published experimental data!
    Application Design example Length Target Ref.
    Gene Silencing mRNA 5’-NNNnnnnnnnnNNN-3’
    5′-NNnnnNNNnNnnnnnNnNNn-3′
    14mer
    22mer
    Yamamoto
    et al. 2012
    siRNA 5’-NNNnNnnnnnnnnnnnnnnnn-3’
    5’-nnNnnnNnnnNnnnNnnnNnn-3’
    5’-nnnnnnnnnnNnNNnnnnnnn-3’
    5’-nnNnnNnnnnNnnnnNnnnnn-3’
    21mer Rahman et al. 2008 in J. Am. Chem. Soc. 2008, 130, 4886-4896.
    Antisense 5’-NNnnnnnnnnnnNN-3’ 14mer Prakash et al in 2010.
    Aptamer capping Thrombin binding aptamer was capped at the 3’-end by using BNA-5’-triphosphates and terminal deoxynucleotidyl transferase   Kasahara et al. 2010 in Bioorganic & Medicinal Chemistry Letters 20(2010) 1626-1629.
    Duplex Formation


    Target ssRNA
    5’-d(GCGTTTTTTGCT)-3’
    5’-d(GCGTTTTTTGCT)-3’
    5’-d(GCGTTTTTTGCT)-3’
    5’-r(AGCAAAAACGC)-3’
    Tm = 50
    Tm = 59
    Tm = 80
    Rahman et al. 2005 in Nucleic Acids Symposium Series No. 48 pp 5-6
    Duplex Formation

    Target ssRNA
    5’-d(GCGTTYTTTGCT)-3’
    5’-d(GCGYTYTYTGCT)-3’
    5’-r(AGCAAAAACGC)-3’
    Y = 2’,4’-BNANC [NMe] thymine monomer
    Tm = 50
    Tm = 63
    Rahman et al. 2007 in Nucleosides, Nucleotides, and Nucleic Acids, 26:1625–1628, 2007
    Selective Duplex Formation with ssRNA
    5’-d(GCGTTTTTGCT)-3’(7)
    5’-d(GCGTTTTTTGCT)-3’(8)
    5’-d(GCGTTTTTTGCT)-3’(9)

    Change in melting temperature (ΔTm) of modified oligonucleotides relative to 5’-d(GCGTTTTTTTGCT)-3’
    Conditions: 4 mM strands solution in 10 mM sodium phosphate buffer (pH 7.2) containing 100 mM NaCl.
    Target DNA, 5’-d(AGCAAAAAACGC)-3’
    Target RNA, 5’-r(AGCAAAAAACGC)-3’
      Miyashita et al. Chem. Commun., 2007, 3765–3767.
    Duplex Formation
    Target ssRNA
    Target ssDNA
    5-d(GCGTTTTTTGCT)
    5’-r(AGCAAAAAACGT)-3’, Tm = 63
    5’-r(AGCAAAAAACGT)-3’, Tm = 51
    12mer Rahman et al. 2008 in J. Am. Chem. Soc. 2008, 130, 4886-4896.
    Triplex Formation

    Target dsDNA
    5’-d(TTTTTmCTYTmCTmCTmCT)-3’
    5’-d(GCTAAAAAGAAAGAGAGATCG)-3’
    3’-d(CGATTTTTCTTTCTCTCTAGC)-5’
    Y = 2’,4’-BNANC [NMe] thymine monomer, mC = 5-methylcytidine
    Tm = 38 Rahman et al. 2007 in Nucleosides, Nucleotides, and Nucleic Acids, 26:1625–1628, 2007
    Triplex Formation(TFO) dsDNA 5-d(TTTTTCTTTCTCT)

    5’-GCTAAAAAGAAAGAGAGATCG)-3’
    3’-CGATTTTTCTTTCTCTCTCTAGC)-5’

    5’-TTTTTmCTTTmCTmCTmCT-3’
    5’-TTTTTmCTTTmCTmCTmCT-3’
    5’-TTTTTmCTTTmCTmCTmCT-3’

    5’-d(GCTAAAAAGAAAGAGAGATCG)-3’
    3’-d(CGATTTTTCTTTCTCTCTAGC)-5’





    Tm = 59
    Tm = 57
    Tm = 65
    Rahman et al. 2008

    Rahman et al. 2007
    in Angew. Chem. Int. Ed. 2007, 46,4306-4309.
    PCR clamping with BNA oligos The exact ratio of BNA to DNA monomers is dependent on the target Tm value, the length of the oligo, GC%, and target sequence. In general, more than 50% of a15-mer DNA oligo should be modified by BNA-NC units and contain a phosphate group at the 3’-end. BNA oligos bind more effectively than LNA or PNA oligos.  
    Telomer Fish Probes      
    telomere repeat sequence 5’-CCCTAACCCTAACCCTAA-3’ Telomers  
    major satellite repeats 5’-TCGCCATATTCCAGGTC-3’ Centromeres  
    (TTAGG)4 repeat 5’-TTAGGTTAGGTTAGG-3’ Repeats  
    (TTAGGG)n repeat 5’-TTAGGGTTAGGGTTAGGG-3’ Repeats  
    (CCCTTA)n repeat 5’-CCCTTACCCTTACCCTTA-3’ Repeats  
    repetitive hexameric sequences 5’-TTAGGGTTAGGGTTAGGG-3’ distal end of chromosomes  

    N = position of BNA NC [NMe] monomer.

    Probes are usually labeled with Cy3 or FITC at the 5’ or 3’ end but other dyes may be used as well.
    Concentrations of artificial oligonucleotides and buffers used for BNA experiments by Imanishi and others!

    Application Concentration of oligonucleotide per strand Buffer Reference
    Antisense BNAs 1 micomolar (1 uM) Buffer: 20 mM sodium phosphate pH 7.2 Obika et al., 2001
    Duplex formation 4 micromolar (uM) 100 mM NaCl, 10 mM sodium phosphate pH 7.2 Obika et al., 1998
    Duplex formation 4 microM (uM) 100 mM NaCl, 10 mM sodium phosphate pH 7.2 Imanishi and Obika 2002
    Duplex formation 4 micromolar (uM) 100 mM NaCl, 10 mM sodium phosphate pH 7.2 Miyashita et al., 2007
    Triplex formation   140 mM KCl, 10 mM MgCl2, 7 mM sodium phosphate pH 7.0 Hari et al., 2003

     

    More details on design and experimental results:

    1. Gene silencing by targeting mRNA usingBNANC[NMe]’s !

    Design of BNANC oligonucleotide: 5’-NNNnnnnnnnnNNN-3’,


    where N depicts the position of a BNAnc monomer, and n depicts the position of a natural DNA. The use of BNANC’s increased nuclease resistance and improved overall properties for gene silencing by targeting mRNA. Buffer used: 100 mM NaCl, 10 mM sodium phosphate pH 7.2 for thermal melting studies. In vitro transfection was done with Lipofetamine 2000.

    2′,4′-BNANC–based AONs are promising therapeutic agents for antisense therapy!! BNANC’s for gene silencing by mRNA targeting!

    Yamamoto et al. 2012 showed that 2’,4’-BNANC-based AONs targeting apoB mRNA have higher binding affinities to the target RNA than do 2’,4’-BNA/LNA-based AONs. Additionally, in vitro transfection studies revealed the superior silencing effect of short 2’,4’-BNANC-based AONs (<20-ntlong), indicating that 2’,4’-BNANC may have advantageous properties as short antisense drugs. The BNANCs corresponding to the LNAs showed stronger inhibitory activities. Shorter AONs (13- to 14mers) showed better inhibitory activities. The 2’,4’-BNANC-14mer worked the best.

    2. Gene silencing by targeting mRNA!

    Design of BNANC oligonucleotide: 5′-NNnnnNNNnNnnnnnNnNNn-3′


    Yamamoto et al. 2012: In vitro and in-vivo study. BNANC[NMe]’s with cholesterol lowering action targeting PCSK9 and with increased nuclease resistance and improved properties for gene silencing by targeting mRNA. Yamamoto and colleges achieved a dose-dependent decrease in serum LDL-C levels by using a 2′,4′-BNANC–based AON (P901SNC). Serum HDL-C levels and the levels of liver and kidney toxicity indicators were not elevated. Histopathological analysis revealed no severe hepatic toxicities. They also showed that a 2′,4′-BNANC–based AON (P901SNC) has greater potential to inhibit PCSK9 and to reduce serum cholesterol levels with no toxicity than a conventional 2′,4′-BNA–based AON. The high-potency and low-toxicity characteristics of a 2′,4′-BNANC–based AON were previously reported to effectively inhibit PTEN mRNA without elevation of the serum ALT level, whereas elevated serum ALT was observed in the 2′,4′-BNA counterpart-treated arm. Thus, it was concluded that 2′,4′-BNANC–based AONs can be a promising therapeutic agent for antisense therapy.

    3. Interference RNA: => siRNA: BNANC’s for siRNA - RNAi => siRNA

    Design of BNANC oligonucleotides that worked best:


    5’-NNNnNnnnnnnnnnnnnnnnn-3’,
    5’-nnNnnnNnnnNnnnNnnnNnn-3’
    5’-nnnnnnnnnnNnNNnnnnnnn-3’,
    5’-nnNnnNnnnnNnnnnNnnnnn-3’

    Rahman and colleges in 2010 found that 2’,4’-BNA- and 2’,4’-BNANC-modified siRNAs are equally compatible with the RNAi machinery similar to that observed for natural siRNA. To improve siRNA biostability, a number of bridged nucleotide moieties can be incorporated in the sense strand without loss of the usual gene silencing property. Thermally stable functional siRNAs can also be obtained by slightly modifying the middle of the sense and antisense strands together. Unlike the 3’-overhang modification, this modification increased Tm satisfactorily and contains an antisense strand with BNA residues which might be more efficacious in gene silencing. Modification at the Ago2 cleavage site (9–11th positions) produced variable results based on siRNA composition and sequence; usually the modification at the 10th position of the sense strand is more sensitive. Modification at the 11th position of the cleavage site is safer than that of the 10th or 9th position. For the first time, this study as a whole shows the utility and capability of 2’,4’-BNANC, a highly stable and efficient nucleic acid derivative in RNAi technology, and also gives some new ideas about designing biostable, functional siRNAs consisting both of 2’,4’-BNA and 2’,4’-BNANC residues.

    4. Antisense oligonucleotide targeting PTEN mRNA

    Design of BNANC oligonucleotide:


    5’-NNnnnnnnnnnnNN-3’ 14mer
    43 d(CUTAGCACTGGCCU)30 20,40-BNANC[NMe] PTEN
    Prakash et al in 2010.


    5. Aptamer Capping at the 3’-end

    A thrombin aptamer was capped with BNAs at the 3’-end. This increased nuclease resistance and the stability of the aptamer. Kasahara et al. 2010.

    6. Synthesis of Novel 2’,4’-BNANC [NMe] nucleic acid analog.

    Duplex formation: 5-d(GCGTTTTTTGCT)

    Target ssRNA 5’-r(AGCAAAAAACGT)-3’, Tm = 63

    Target ssdNA 5’-r(AGCAAAAAACGT)-3’, Tm = 51

    Triplex formation: 5-d(TTTTTCTTTCTCT) 2’,4’-BNANC [NH] and LNA are better.
    Target: dsDNA 5’-GCTAAAAAGAAAGAGAGATCG)-3’
    3’-CGATTTTTCTTTCTCTCTCTAGC)-5’
    Rahman et al. 2008.
    A list of rules for the design of probes using BNAs

     

    BNAs
    (i) Place one BNA monomer per every 3 bases within an oligomer.
    For example in a 20mer oligomer, about 4 BNA monomers can be inserted (there is some degree of freedom as to the exact positions, in other words, they do not have to be exactly every 3 to 6 bases).
    According to the specific application, the mode of BNA modification may need to be changed. Both gapmer- and chimera-modification with BNA-NC will be effective for antisense application. More than four continuous natural DNA monomers as part in a BNA-modified oligonucleotide was found to be necessary to recruit RNase H. For diagnostic application, the modification of one bp with a BNA nucleotide maybe enough in some cases.
    (ii) The spacing of the BNA monomers need not be different within the oligo for different applications. For example, if the spacings are appropriate the same monomer may be used.
    (iii) The use of no more than 4-8 BNA’s within a 20 mer probe is recommended but is depended on the specific application.
    (iv) Each BNA –NC monomer increase the Tm by about 4 degrees Celsius. Therefore this can be used to estimate the Tm of the BNA containing oligonucleotide). I agree.
    (v) An oligonucleotide designed with 2’, 4’-BNA-NC[NH] is more selective to ssRNA and binds more strongly than LNA if more bases are modified.
    (vi) An oligonucleotide designed with 2’, 4’-BNA-NC[NH] binds also to ssDNA and binds also more strongly than LNA if more bases are modified. The ssDNA binding strength of BNA-NC(NH) is equivalent to (or slightly more than) that of LNA.
    (vii) BNA-NC(NMe) modification add a very high nuclease-resistance to the oligonucleotide (much more than LNA modification). This property is desirable not only for therapeutic application but also for diagnostic use.
    References for the basic properties and some applications for BNA-NC:
    Chem. Commun., 2007, 3765;
    Angew. Chem. Int. Ed., 2007, 46, 4306;
    J. Am. Chem. Soc., 2008, 130, 4886;
    Bioorg. Med. Chem., 2010, 18, 3473;
    Molecular Therapy NA, 2011, 1, e22;
    J. Nucleic Acids, 2012, ID 707323.

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    Rules for the design of allele specific primers

    For regular DNA primers: Design primers that are specific to the 3’ end nucleotide of the SNP and the 3rd nucleotide from the 3’ end is mismatched with the template DNA.
    Design primers that are specific to the 2nd nucleotide from the 3’ end of the SNP and the 3rd nucleotide from the 3’ end is mismatched with the template DNA.

    To avoid the “pseudopositive problem”: Figure 2 illustrates concepts in the rational design of biological active peptides and peptidomimetics.
    Design primers that are specific to the 3’ nucleotide of the SNP and the other two nucleotides located downstream from the location of the SNP are mismatched with the template DNA.

    For BNA based primers: Add one BNA/LNA monomer per primer at the 3’ end where the SNP is located.

    Resources:

    To address the“pseudopositive problem” and to minimize the “pseudopositive” signal the primers were designed by Yaku et al. in 2008 with a 3’ nucleotide matching the template plus 2 mismatch nucleotides:

    Yaku et al. in 2008 showed that selecting a primer having a 3’ terminal nucleotide that recognizes the SNP nucleotide and the next two nucleotides that form mismatch pairings with the template sequence can be used as an allele-specific primer to eliminate the pseudopositive problem.

    Using primers for the human ABO genes the researchers demonstrated that this primer design is also useful for detecting a single base pair difference in gene sequences with a signal-to-noise ratio of at least 45.

    However, the literature citing experimental data indicates that proper design of primer DNA sequences is important for the efficient detection of SNP by PCR. Allele-specific primers are usually designed to complement template DNA and contain a nucleotide specific to the SNP at the 3’ end.

    The SNP-specific nucleotide forms a base pairing or mismatch pairing depending on the base pair identity of the SNP. Only a proper base pairing at the end of the primer/template duplex will produce a PCR product. Less PCR products are produced for terminal mismatch pairings due to decreased DNA polymerase binding and inefficiencies in the incorporation of 2’-deoxyribonucleoside triphosphates. However, undesired primer extension with mismatched DNA primers can occur during the PCR reaction under unsuitable reaction conditions when the proper amplification cycle, reaction time, temperature, and 2’-deoxyribonucleoside triphosphate concentrations are not optimized. This is called the “pseudopositive problem”, that can also arise due to specific DNA primer sequences. Primer extension reactions are often observed when a single mismatch is formed at the 3’ end with the primer. When SNP typing is used to distinguish homozygotes and heterozygotes this problem becomes more serious since the pseudopositive signal should be less than 1% of those obtained with matched primers. Therefore allele-specific primers have been designed so that the 3’ end nucleotide was specific to the SNP and the 3rd nucleotide from the 3’ end was mismatched with the template DNA (Kambara and coworkers 2004). Another research group developed primers so that the 2nd nucleotide from the 3’ end was specific to the SNP and the 3rd nucleotide from the 3’ end was mismatched with the template DNA (Aono et al., 2000). Methods using locked nucleic acids (LNA), phosphorothioate-modified primer, and dideoxynucleotide-terminated primer have been reported.

    Example Template DNA Forward primer Reverse primer
    lambda DNA 5’-GATGAGTTCGTGTCCGTACAACX3X2X1-3’ Complementary to base pairs 7131–7155 of the lambda DNA sequence, forming zero, one, two, or three mismatch pairings at the 3’ end depending on the identity of X1, X2, and X3 = BNA monomer. 5’-GAATCACGGTATCCGGCTGCGCTGA-3’ Fully matched with base pairs 7406–7430 of the lambda DNA.
    Using BNAs 5’-GATGAGTTCGTGTCCGTACAACX
    X = BNA monomer. = Placement of BNA monomer
     

    Example for the location of primers for lambda DNA:

    Sequence from Enterobacteria phage HK629, complete genome:

    Variants Forward Primer

    GATCCGGCGCGTGAGTTCACCATGATTCAGTCAGCACCGCTGATGCTGCTGGCTGACCCTGATGAGTTCGTGTCCGTACAACTGG 5’-GATGAGTTCGTGTCCGTACAACX3X2X1
    3’-CTACTCAAGCACAGGCATGTTGACC
    Taq Polymerase ->

    X1 = A, T, or C; X2X3 = CA, CT, CC, AA, AT, AC, TA, TT, OR TC

    CGTAATCATGGCCCTTCGGGGCCATTGTTTCTCTGTGGAGGAGTCCATGACGAAAGATGAACTGATTGCCCGTCT
    CCGCTCGCTGGGTGAACAACTGAACCGTGATGTCAGCCTGACGGGGACGAAAGAAGAACTGGCGCTCCGTGTGGCAGAGC
    TGAAAGAGGAGCTTGATGACACGGATGAAACTGCCGGTCAGGACACCCCTCTCAGCCGGGAAAATGTGCTGACCGGACAT
    GAAAATGAGGTGGGATCAGCGCAGCCGGATACCGTGATTCTGGATACGTCTGAACTGGTCACGGTCGTGGCACTGGTGAA
    3’-AGTCGCGFCGGCCTATGGCACTAAG-5’
    <- Taq Polymerase
    5’-GAATCACGGTATCCGGCTGCGCTGA-3’
    Reverse Primer
    A single amplification product should be observed corresponding to the amplified region of the DNA.

    Primers for ABO genotyping using exon 6 of the ABO gene

    www.ncbi.nlm.nih.gov/books/NBK2267/
    Sequences of exon 6 of the ABO gene
    ABO gene Sequence (5’-3’) (Underlined nucleotides, the G and the A, are the 22 nd nucleotide in the AB allele and the O allele, respectively).

    A allele
    1 TAGGAAGGAT GTCCTCGTGG TGACCCCTTG GCTGGCTCCC
    B allele
    41 ATTGTCTGGG AGGGCACATT CAACATCGAC ATCCTCAACG
    81 AGCAGTTCAG GCTCCAGAAC ACCACCATTG GGTTAACTGT
    121 GTTTGCCATC AAGAA
    O allele
    1 TAGGAAGGAT GTCCTCGTGG TACCCCTTGG CTGGCTCCCA
    41 TTGTCTGGGA GGGCACATTC AACATCGACA TCCTCAACGA
    81 GCAGTTCAGG CTCCAGAACA CCACCATTGG GTTAACTGTG
    121 TTTGCCATCA AGAA

    The selected target for human blood genotyping was the 22nd base pair in exon 6 of the ABO. This base pair is a G/C base pair in the A and B alleles and an A/T base pair in the O allele due to deletion of the G/C base pair found in the A and B alleles.

    The 22ndbase pair in exon 6 is the homo G/C base pair for blood type AB and the homo A/T base pair for blood type O.

    A two-step PCR was carried out to detect allelic difference in the 22nd base pair of exon 6 of the ABO gene.

    1st PCR:

    A fragment of exon 6 in human genomic DNA was amplified by using a forward primer (5’-TAGGAAGGATGTCCTCG-3’) complementary to base pairs 1–17 and a reverse primer (5’-TTCTTGATGGCAAACACAGTTAAC-3’) complementary to base pairs 112–135 of the A and B alleles or base pairs 111–134 of the O allele on exon 6.
    PCR reaction was carried out in 20 µL reactions using the LightCycler FastStart DNA Master SYBR Green I reaction kit with 0.5 ng/mL of genomic DNA, 1.25 mM MgCl2, and 1 mM of each of the forward and reverse primers. Following denaturation at 950C for 10 min, 50 cycles of denaturation at 950C for 10 seconds, annealing at 520C for 10 seconds, and extension at 720C for 10 seconds were carried out on the LightCycler. Amplification was monitored in real time by measuring the fluorescent intensity of SYBR Green I, and amplifications were confirmed to be completed by the 50th cycle with the amount of amplification product being almost identical for both alleles.

    2nd PCR:

    ABO genotyping with allele-allele-specific primers was carried out using the product of the 1st PCR as a template and the allele-specific forward primers given in Table 3 (5’-TAGGAAGGATGTCCTCGTGY3Y2G-3’) in the 2nd PCR.
    The 3’ end nucleotide of the primers is G, which is complementary to the C of the 22nd G/C base pair of exon 6 in the A and B alleles but is mismatched with the A/T base pair at base pair 22 of the O allele. The 2nd and 3rd nucleotides from the 3’ end, Y2 and Y3, respectively, are designed to be mismatched with the 20th and 21st nucleotides of the exon 6 sequence.
    The reverse primer used for the 2nd PCR was 5’-TTCTTGATGGCAAACACAGTTAACC-3’. The PCR mixtures (20 µL) contained 2 µL of the 1st PCR product diluted 1000-times, 1.25mM MgCl2 , 1mM of each of the forward and reverse primers.
    Following DNA denaturation at 950C for 10 min, the 2nd PCR was carried out for 21–28 cycles of denaturation at 950C for 10 seconds, annealing at 520C for 10 seconds, and extension at 720C for 10 seconds.
    Isolated-genomic-DNA

    Schematics of the detection of a single base pair difference in exon 6 of the ABO gene.

    (Source: Yaku et al., 2008)

    Allele-specific forward primers used for the detection of single base pair difference in the AB allele and the O allele:
    Allele’ specific primer Sequence (5’-3’)a)
    ABO261’ AAG TAGGAAGGATGTCCTCGTGAAG
    ABO261’ ACG TAGGAAGGATGTCCTCGTGACG
    ABO261’ AGG TAGGAAGGATGTCCTCGTGAGG
    ABO261’ CAG TAGGAAGGATGTCCTCGTGCAG
    ABO261’ CCG TAGGAAGGATGTCCTCGTGCCG
    ABO261’ CGG TAGGAAGGATGTCCTCGTGCGG
    ABO261’ TAG TAGGAAGGATGTCCTCGTGTAG
    ABO261’ TCG TAGGAAGGATGTCCTCGTGTCG
    ABO261’ TGG TAGGAAGGATGTCCTCGTGTGG
    a) Underlined nucleotides are unpaired with the AB allele and the O allele

    PCR

    After initial denaturation at 950C for 10 min, the amplification was carried out for 20 or 30 cycles as follows in a LightCycler (Roche Diagnostics) thermal cycler: denaturation at 95ºC for 10 seconds, annealing at 58ºC for 10 seconds, and DNA extension at 720C for 10 seconds. The 20-µL PCR mixtures were prepared using the LightCycler FastStart DNA Master SYBR Green I reaction kit (Roche Diagnostics, with 1 ng/mL lambda DNA, 1.25mM MgCl2, 1 mM forward primer, and 1 mM reverse primer. PCR products were analyzed by electrophoreses on 3% agarose gel.

    SNP info:

    Single nucleotide polymorphisms (SNPs) are gene polymorphisms that occur at circa 0.1% in the human genome, and more than three million SNP have been identified so far. Several associations of SNP types with diseases including diabetes, cancer, and myocardial infarction, and SNP in the human genome. SNPs are also known to influence other aspects of human health such as blood group type and the sensitivity to alcohol. Several techniques for SNP genotyping are available.

    These include:
    1. DNA hybridization,
    2. Primer extension reaction using allele-specific DNA primers
    3. The use of DNA polymerase
    4. The use of DNA mismatch-recognizing enzymes
    5. The Invader assay
    6. DNA chips
    7. Pyrosequencing
    The use of allele-specific primers have the advantage to be efficient in cost, reaction time, and simplicity of handling.
    SNP genotyping can be achieved by detecting the amounts of PCR products or by detecting the pyrophosphate generated during PCR depending on the identities of the base pairs in the SNP and the template DNA.

    References

    Hidenobu Yaku, Tetsuo Yukimasa, Shu-ichi Nakano, Naoki Sugimoto, Hiroaki Oka;Design of allele-specific primers and detection of the human ABO genotyping to avoid the pseudopositive problem Electrophoresis 2008, 29, 4130–4140.

    Designed primers that are specific to the 3’ nucleotide of the SNP and the other two nucleotides located downstream from the location of the SNP are mismatched with the template DNA.

    Ophélia Maertens, Eric Legius, Frank Speleman, Ludwine Messiaen, Jo Vandesompele,¤Real-time quantitative allele discrimination assay using 3’-locked nucleic acid primers for detection of low-percentage mosaic mutations. Analytical Biochemistry 359 (2006) 144–146.
    Six AS-PCR reverse primers were evaluated for their discriminating power: a wild-type or mutant DNA primer with an additional 3’ subterminal mismatch (underlined) (5’-CTAGTTTGGTCTGGGCTTGTTG/A-3’) and two wild-type or mutant 3’ LNA (bold) primers (5’-CTAGTTTGGTCTGGGCTTGTCG/A-3’ and 5’-CTAGTTTGGTCTGGGCTTGTTG/A-3’).
    Amplification

    Fig.1.Amplification plots of mutant plasmid using different AS-PCR Primers: mutant (rectangle) and wild-type (triangle) DNA primer(green), 3'LNA primer (blue),and 3' LNA primer with 3' sub terminal mismatch (red)(replicates are show).(For interpretation of the references to color in this figure legend, the reader is referred to the web version of this)
    Luca Morandi, Dario de Biase, Michela Visani, Valentin; Allele Specific Locked Nucleic Acid Quantitative PCR (ASLNAqPCR): An Accurate and Cost-Effective Assay to Diagnose and Quantify KRAS and BRAF Mutation. PLoS ONE | www.plosone.org 1 April 2012 | Volume 7 | Issue 4 | e36084.
    Morandi et al. report the design of a novel assay that was called“Allele Specific Locked Nucleic Acid quantitative PCR” (ASLNAqPCR). The assay uses LNA-modified allele specific primers and LNA-modified beacon probes to increase sensitivity, specificity and to accurately quantify mutations. Primers specific for codon 12/13 KRAS mutations and BRAF V600E were designed and the assay was validated with 300 routine samples from a variety of sources, including cytology specimens.

    Primers used:

    Sanger Sequencing
    Gene Exon Forward Primer Reverse Primer
    KRAS 2 AAGGTGAGTTTGTATTAAAAGGTACTGG TGGTCCTGCACCAGTAATATGC
      3 TCCAG AC TGT GTTTCT CCCTTCTC AA AA CTATAA TT A CTCCTTAA TG TCAG CTT
    8RAF 15 TCATAATGCTTGCTCTGATAGGA GGCCAAAAATTTAATCAGTGGA
    ASLNAqPCR
    Gene WT/Mutation Forward Primer Forward Primer
    KRAS WT GGTAGTTGGAGCTGGTGGC AGAGTGCCTTGACGATACA
      G12A TGTGGTAGTTGGAGCTG+C AGAGTGCCTTGACGATACA
      G12C CTTGTGGTAGTTGGAGCT+T AGAGTGCCTTGACGATACA
      G12D GTGGTAGTTGGAGCTG+A AGAGTGCCTTGACGATACA
      G12R CTTGTGGTAGTTGGAGCT+C AGAGTGCCTTGACGATACA
      G12S TTGTGGTAGTTGGAGCT-tA AGAGTGCCTTGACGATACA
      G12V TTGTGGTAGTTGGAGCTGtT AGAGTGCCTTGACGATACA
      G130 GTAGTTGGAGCTGGTG+A AGAGTGCCTTGACGATACA
      MACON 5'- FA M-CCGGTG AAG A+GT-tGCCTTG A-tCG ATA+CAGCACCGG- BH -3'
    8RAF WT TAG G TG ATTTTG G TCTA GCTA CAG+T TT AA TCAG TG G AA AAA TAG CCTCA
      V600E TAG G TG ATTTTG G TCTA GCTA CAG+A TT AA TCAG TG G AA AAA TAGCCTCA
      BEACON 5'-FAM^CGAAGGGGATC-»-CAGACAA-t-CTGTTCAAACTGCCTTCGG-3BHQ-1 -3
    bp, base pair. precedes INA-modified nucleotides.
    do«:10.1371/journal.pone.0036084.t003

    Schematics of the Assay

    illustrating

    Figure 1. Diagram illustrating ASLNAqPCR.Left side:a single mismatch of the modified primer does not allow PCR amplification.Right side:in case of a perfect match, the Taq polymerase extends the DNA stand and the amplicon is detected by the interrnal LAN modified beacon probe.
    doi:101371/journal.pone.0036084.g001
    Joy Nakitandwe, Friederike Trognitz and Bodo Trognitz; Reliable allele detection using SNP-based PCR primers containing Locked Nucleic Acid: application in genetic mapping. Plant Methods 2007, 3:2 doi:10.1186/1746-4811-3-2.
    The research group presents “a fast and cost-effective protocol for the detection of allele-specific SNPs by applying Sequence Polymorphism-Derived (SPD) markers. These markers proved highly efficient primers for fingerprinting of individuals possessing a homogeneous genetic background. SPD markers are obtained from within non-informative, conventional molecular marker fragments that are screened for SNPs to design allele-specific PCR primers. The method makes use of primers containing a single, 3'-terminal Locked Nucleic Acid (LNA) base. They “demonstrate the applicability of the technique by successful genetic mapping of allele-specific SNP markers derived from monomorphic Conserved Ortholog Set II (COSII) markers mapped to Solanum chromosomes, in S. caripense. By using SPD markers it was possible for the first time to map the S. caripense alleles of 16 chromosome-specific COSII markers and to assign eight of the twelve linkage groups to consensus Solanum chromosomes. The conclusion: The method based on individual allelic variants allows for a level-of-magnitude higher resolution of genetic variation than conventional marker techniques”. They showed “that the majority of monomorphic molecular marker fragments from organisms with reduced heterozygosity levels still contain SNPs that are sufficient to trace individual alleles”.

     


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    Heterogeneous nuclear ribonucleoproteins (hnRNPs) isoforms

    Heterogeneous nuclear ribonucleoproteins (hnRNPs) comprise a family of RNA-binding proteins. The nature of hnRNPs is very complex and diverse. This multifunctional protein family is involved not only in processing heterogeneous nuclear RNAs (hnRNAs) into mature mRNAs, but also acting as trans-factors in regulating gene expression. Heterogeneous nuclear ribonucleoprotein E1 (hnRNP E1), a subgroup of hnRNPs, is a KH-triple repeat containing the RNA-binding protein. It is encoded by an intronless gene arising from hnRNP E2 through a retrotransposition event. hnRNP E1 is ubiquitously expressed and functions in regulating major steps of gene expression, including pre-mRNA processing, mRNA stability, and translation. This protein family exhibit wide-ranging functions in the nucleus and cytoplasm and interaction with multiple other proteins and appears to be involved in post-transcriptional regulation events or pathways in eukaryotic organisms.

    The A/B subfamily of ubiquitously expressed hnRNPs

    This gene belongs to the A/B subfamily of ubiquitously expressed heterogeneous nuclear ribonucleo-proteins (hnRNPs). The hnRNPs are RNA binding proteins and complex with heterogeneous nuclear RNA (hnRNA). These proteins are associated with pre-mRNAs in the nucleus and appear to influence pre-mRNA processing and other aspects of mRNA metabolism and transport. While all of the hnRNPs are present in the nucleus, some seem to shuttle between the nucleus and the cytoplasm. The hnRNP proteins have distinct nucleic acid binding properties. The protein encoded by this gene has two repeats of quasi-RRM domains that bind to RNAs. It is one of the most abundant core proteins of hnRNP complexes and it is localized to the nucleoplasm. This protein, along with other hnRNP proteins, is exported from the nucleus, probably bound to mRNA, and is immediately re-imported. Its M9 domain acts as both a nuclear localization and nuclear export signal. The encoded protein is involved in the packaging of pre-mRNA into hnRNP particles, transport of poly A+ mRNA from the nucleus to the cytoplasm, and may modulate splice site selection. It is also thought to have a primary role in the formation of specific myometrial protein species in childbirth. [The myometrium is the middle layer of the uterine wall, mainly consisting of uterine smooth muscle cells.] Multiple alternatively spliced transcript variants have been found for this gene but only two transcripts are fully described. These variants have multiple alternative transcription initiation sites and multiple polyA sites. [From Refseq: http://www.ncbi.nlm.nih.gov/refseq/].

    Precursor mRNA

    An immature single strand of messenger ribonucleic acid (mRNA) is called a precursor mRNA (pre-mRNA) which is synthesized in the cell nucleus from a DNA template by transcription. Pre-mRNA makes up the majority of heterogeneous nuclear RNA (hnRNA). hnRNA can also include RNA transcripts that do not end up as cytoplasmic mRNA. More details about these proteins can be found at http://atlasgeneticsoncology.org/Genes/GC_HNRNPA1.html and http://www.uniprot.org/uniprot/P09651.
    The heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is sometimes also called “Helix-destabilizing protein”, “Single-strand RNA-binding protein”, or “hnRNP core protein A1” and was identified in the spliceosome C complex, and in a mRNP granule complex, which appears to be at least composed of proteins ACTB, ACTN4, DHX9, ERG, HNRNPA1, HNRNPA2B1, HNRNPAB, HNRNPD, HNRNPL, HNRNPR, HNRNPU, HSPA1, HSPA8, IGF2BP1, ILF2, ILF3, NCBP1, NCL, PABPC1, PABPC4, PABPN1, RPLP0, RPS3, RPS3A, RPS4X, RPS8, RPS9, SYNCRIP, TROVE2, YBX1 and untranslated mRNAs. The protein interacts with SEPT6, with HCV NS5B, and with the 5'-UTR and 3'-UTR of HCV RNA. 5′UTR and 3′UTR are the non-coding regions of HCV RNA, referred herein as 5′ and 3′ untranslated regions that contain important sequence and structural elements critical for HCV translation and RNA replication.

    Spliceosome

    The spliceosome is a multimegadalton RNA-protein machine that removes noncoding sequences from nascent pre-mRNAs. Recruitment of the spliceosome to splice sites and subsequent splicing require a series of dynamic interactions among the spliceosome's component U snRNPs and many additional protein factors.

    Pre-mRNA splicing by the U2-type spliceosome

    (A) Schematic representation of the two-step mechanism of pre-mRNA splicing. Boxes and solid lines represent the exons (E1, E2) and the intron, respectively. The branch site adenosine is indicated by the letter A and the phosphate groups (p) at the 5′ and 3′ splice sites, which are conserved in the splicing products, are also shown. (B) Conserved sequences found at the 5′ and 3′ splice sites and branch site of U2-type pre-mRNA introns in metazoans and budding yeast (S. cerevisiae). Y = pyrimidine and R = purine. The polypyrimidine tract is indicated by (Yn). (C) Canonical cross-intron assembly and disassembly pathway of the U2-dependent spliceosome. For simplicity, the ordered interactions of the snRNPs (indicated by circles), but not those of non-snRNP proteins, are shown. The various spliceosomal
    complexes are named according to the metazoan nomenclature. Exon and intron sequences are indicated by boxes and lines, respectively. The stages at which the evolutionarily conserved DExH/D-box RNA ATPases/helicases Prp5, Sub2/UAP56, Prp28, Brr2, Prp2, Prp16, Prp22 and Prp43, or the GTPase Snu114, act to facilitate conformational changes are indicated. (D) Model of interactions occurring during exon definition. (From: Will C L , and Lührmann R Cold Spring Harb Perspect Biol 2011;3:a003707).

    The DExH/D protein family

    The DExH/D protein family is the largest group of enzymes found in eukaryotic RNA metabolism. DExH/D proteins unwind RNA duplexes in an ATP-dependent fashion. In recent years it has become clear that these DExH/D RNA helicases are also involved in the ATP-dependent remodeling of RNA–protein complexes. DExH/D proteins are essential for all aspects of cellular RNA metabolism and processing, for the replication of many viruses and for DNA replication as well. The DExH/D protein family database contains information about these proteins and makes it available over the WWW (http://www.columbia.edu/~ej67/dbhome.htm).

    Exon definition

    During exon definition exons are recognized and defined as units during early assembly by binding of factors to the 3' end of the intron, followed by a search for a downstream 5' splice site. The presence of both a 3' and a 5' splice site in the correct orientation and within 300 nucleotides of one another will allow that stable exon complexes are formed. Concerted recognition of exons may help explain the 300-nucleotide-length maximum of vertebrate internal exons, the mechanism whereby the splicing machinery ignores cryptic sites within introns, the mechanism whereby exon skipping is normally avoided, and the phenotypes of 5' splice site mutations that inhibit splicing of neighboring introns (Roberson et al. 1990).

    Cytoplasmic mRNP granules at a glance

    (Source: Stacy L. Erickson and Jens Lykke-Andersen; Cytoplasmic mRNP granules at a glance Journal of Cell Science 124, 293-297).
    Eukaryotic gene expression is controlled by translation and mRNA degradation which are important in the regulation of these processes. Translation and steps in the major pathway of mRNA decay are in competition with each other. mRNAs that are not engaged in translation can aggregate into cytoplasmic mRNP granules referred to as processing bodies (P-bodies) and stress granules, which are related to mRNP particles that control translation in early development and neurons. The analyses of P-bodies and stress granules suggest a dynamic process. This process is referred to as the mRNA Cycle, wherein mRNPs can move between polysomes, P-bodies and stress granules although the functional roles of mRNP assembly into higher order structures remain poorly understood. (Source: Carolyn J. Decker and Roy Parker; P-Bodies and Stress Granules: Possible Roles in the Control of Translation and mRNA Degradation. Cold Spring Harb Perspect Biol a012286 First published online July 3, 2012).

    Structure of the Heterogeneous nuclear ribonucleoprotein A1 isoform [Homo sapiens]

    (Source: pdb 2LYV)
    Chain A, Solution Structure Of The Two Rrm Domains Of Hnrnp A1 (up1) Using Segmental Isotope Labeling

    PubMed Abstract:“Human hnRNP A1 is a multi-functional protein involved in many aspects of nucleic-acid processing such as alternative splicing, micro-RNA biogenesis, nucleo-cytoplasmic mRNA transport and telomere biogenesis and maintenance. The N-terminal region of hnRNP A1, also named unwinding protein 1 (UP1), is composed of two closely related RNA recognition motifs (RRM), and is followed by a C-terminal glycine rich region. Although crystal structures of UP1 revealed inter-domain interactions between RRM1 and RRM2 in both the free and bound form of UP1, these interactions have never been established in solution. Moreover, the relative orientation of hnRNP A1 RRMs is different in the free and bound crystal structures of UP1, raising the question of the biological significance of this domain movement. In the present study, we have used NMR spectroscopy in combination with segmental isotope labeling techniques to carefully analyze the inter-RRM contacts present in solution and subsequently determine the structure of UP1 in solution. Our data unambiguously demonstrate that hnRNP A1 RRMs interact in solution, and surprisingly, the relative orientation of the two RRMs observed in solution is different from the one found in the crystal structure of free UP1 and rather resembles the one observed in the nucleic-acid bound form of the protein. This strongly supports the idea that the two RRMs of hnRNP A1 have a single defined relative orientation which is the conformation previously observed in the bound form and now observed in solution using NMR. It is likely that the conformation in the crystal structure of the free form is a less stable form induced by crystal contacts. Importantly, the relative orientation of the RRMs in proteins containing multiple-RRMs strongly influences the RNA binding topologies that are practically accessible to these proteins. Indeed, RRM domains are asymmetric binding platforms contacting single-stranded nucleic acids in a single defined orientation. Therefore, the path of the nucleic acid molecule on the multiple RRM domains is strongly dependent on whether the RRMs are interacting with each other.” (Taken from: http://www.rcsb.org/pdb/explore/explore.do?structureId=2LYV).

    The alignment of 3 hnRNP A1 protein sequences is shown below

    Protein and Peptide Library Information for hnRNP proteins

    Solid phase peptide synthesis allows the design and creation of libraries compromising a collection of synthetic peptides. Synthetic libraries have been and can be successfully used for antibody epitope mapping, the determination of the specificity of antibodies, identification of bioactive peptides, development of biological assays, T-cell epitope mapping, vaccine efficacy testing, the screening for ligand-binding activities, screening for antimicrobial peptide activities, peptide-protein interactions, drug discovery, for LC-MS/MS method development and validation as well as for receptor-ligand studies, cellular assays, the study of constrained peptides, modified peptides such as modified histone peptides or the screening for MHC class I and class II peptides, and others.
    Furthermore peptide libraries provide synthetic, crude or purified peptides that can be customized for the development of screening applications such as epitope mapping, phosphorylation site identifications or the identification of other post-translational modification (PTM) sites, peptide target interaction studies, mid- to high-throughput selected reaction monitoring (SRM) and multiple reaction monitoring (MRM) assays in quantitative mass spectrometry (MS) workflows.

    Peptide libraries for proteomics

    The study of proteomes, sub-proteomes and protein pathways often requires quantitative MS analysis that depends on the identification and validation of SRM and MRM assays. Peptide libraries offer great convenience and flexibility in the development of multiple applications involving large numbers of peptides including libraries for quantitative MS approaches. The use of peptide libraries greatly reduces the setup time of MS experiments. The SRMAtlas (http://www.mrmatlas.org/) which attempts to map the entire human proteome can be used as a guide for the development of new types of peptide libraries.

     

    Peptides libraries can be derived from the target protein, and, as an option for the use in MS workflows, with either arginine (R) or lysine (K) as the C-terminal amino acid. Synthetic libraries can cover the most commonly used tryptic proteotypic peptides useful for SRM assay development or proteolytic peptides of the whole protein. The maximum peptide length for screening libraries can range from short peptides (5 amino acids) to 20 amino acids. However, for peptide libraries used in MS workflows the standard maximal length is usually 25 amino acids but this length can be increased up to 35 amino acids to ensure that the vast majority of potential proteotypic peptides that are screened for their suitability for a reliable SRM assay are covered.


    Highlights:
     

    • Convenient– peptides are provided in individual tubes or in 96-well plates
    • Application-specific– C-terminal amino acid of each peptide can be either R or K or any other amino acid
    • Easy to use– peptides can be delivered lyophilized or suspended in 0.1% trifluoroacetic acid (TFA) in 50% (v/v) acetonitrile/water
    • Flexible– extensive list of available modifications
    Includes:

    • Fully synthetic peptides
    • Standard mass spectrometric quality control (QC) analysis
    • Optional modifications, peptide sizes and levels of QC analysis
    • Provided in either in single tubes or 96-well plates

    Example of a peptide library for the hnRNP protein useful for MS workflows

    Hnrnp A1 protein (up1)

    pI of Protein: 8.0, Protein MW: 22246, Amino Acid Composition: A10 C2 D13 E18 F10 G17 H8 I8 K16 L8 M4 N4 P6 Q7 R16 S16 T12 V17 W1 Y4

    1 GGSKSESPKE PEQLRKLFIG GLSFETTDES LRSHFEQWGT LTDCVVMRDP NTKRSRGFGF VTYATVEEVD
      AAMNARPHKV            
    81 DGRVVEPKRA VSREDSQRPG AHLTVKKIFV GGIKEDTEEH HLRDYFEQYG KIEVIEIMTD RGSGKKRGFA
      FVTFDDHDSV            
    161 DKIVIQKYHT VNGHNCEVRK ALSKQEMASA SSSQRGR      

    List of Tryptic Peptides

    #

    m/z
    (mi)

    m/z
    (av)

    Start

    End

    Sequence

    1

    418.2660

    418.5164

    181

    184

    (K)ALSK(Q)

    2

    432.2565

    432.5028

    90

    93

    (R)AVSR(E)

    3

    446.2358

    446.4862

    80

    83

    (K)VDGR(V)

    4

    547.2722

    547.5889

    5

    9

    (K)SESPK(E)

    5

    571.3450

    571.6984

    84

    88

    (R)VVEPK(R)

    6

    574.2831

    574.6147

    49

    53

    (R)DPNTK(R)

    7

    600.4079

    600.7838

    163

    167

    (K)IVIQK(Y)

    8

    733.4607

    733.9346

    108

    114

    (K)IFVGGIK(E)

    9

    771.3995

    771.8533

    10

    15

    (K)EPEQLR(K)

    10

    1049.4575

    1050.1193

    124

    131

    (R)DYFEQYGK(I)

    11

    1165.5232

    1166.1994

    115

    123

    (K)EDTEEHHLR(D)

    12

    1181.5215

    1182.2617

    185

    195

    (K)QEMASASSSQR(G)

    13

    1218.6399

    1219.4513

    132

    141

    (K)IEVIEIMTDR(G)

    14

    1428.6437

    1429.5663

    168

    179

    (K)YHTVNGHNCEVR(K)

    15

    1437.7445

    1438.5928

    94

    106

    (R)EDSQRPGAHLTVK(K)

    16

    1699.7598

    1700.8128

    148

    162

    (R)GFAFVTFDDHDSVDK(I)

    17

    1784.9065

    1786.0038

    17

    32

    (K)LFIGGLSFETTDESLR(S)

    18

    1908.8731

    1910.1941

    33

    48

    (R)SHFEQWGTLTDCVVMR(D)

    19

    2510.2133

    2511.8362

    57

    79

    (R)GFGFVTYATVEEVDAAMNARPHK(V)

    Functional categorization of the proteins for which targeted proteomic assays are available can be found in the MRMAtlas database

    Piccoti et al. in 2008 presented the first database of validated SRM assays for ~1500 yeast proteins. The database was constructed by merging the results of more than 650 SRM-triggered MS2 analyses of S. cerevisiae protein digests, carried out on a triple quadrupole-type mass spectrometer. 1324 proteins are represented by assays for at least one of their peptides proteotypic peptides (PTP’s). The database also contains assays for a small number of peptides common to a maximum of two proteins. The peptides were selected because they show intense signal response by electrospray ionization mass spectrometry. Peptide identifications were validated by collecting a full tandem mass spectrum of the peptides in the QQQ-like mass spectrometer also used for SRM measurements. The database is at present the largest resource of validated SRM/MRM assays of any organism. It currently contains assays for 22% of the yeast proteome and the coverage.
    The database contains assays for yeast proteins involved in all biological processes, as defined by gene ontology (GO) nomenclature. Peptides for proteins spanning all ranges of abundance in yeast are present in the dataset, down to a concentration below 50 molecules/cell.

    The MRMAtlas database

    The dataset can be found in the MRMAtlas (www.mrmatlas.org or www.srmatlas.org) which is publicly accessible. This database
    was created as part of the PeptideAtlas project (www.peptideatlas.org) and can be queried via the web-interface for peptides,
    individual proteins, protein sets, or cellular pathways. These data sets can be used for the design of targeted peptide libraries
    that can be custom synthesized. If desired, all or selected peptides can be labeled with stable isotopes useful for spiking
    experiments.

    Examples of peptide libraries for hnRNP proteins for screening work flows

    A: Rrm Domains of HnRNP A1 (up1): Structure ID: PDB: 2LYV_A

    Sequence in FASTA format

    >gi|433286562|pdb|2LYV|A Chain A, Solution Structure Of The Two Rrm Domains Of Hnrnp A1 (up1) Using Segmental "Isotope Labeling"

    GGSKSESPKEPEQLRKLFIGGLSFETTDESLRSHFEQWGTLTDCVVMRDPNTKRSRGFGFVTYATVEEVDAAMNARPHKVD
    GRVVEPKRAVSREDSQRPGAHLTVKKIFVGGIKEDTEEHHLRDYFEQYGKIEVIEIMTDRGSGKKRGFAFVTFDDHDSVDK
    IVIQKYHTVNGHNCEVRKALSKQEMASASSSQRGR

    Peptide library of 15mers with an overlap of 11 amino acids for HnRNP A1 (up1)

    #

    Peptide

    Position

    Mw

    Chemical Formula

    pI

    Charge

    1

    GGSKSESPKEPEQLR

    1->15

    1628.76

    C67 H113 N21 O26

    7.1

    0

    2

    SESPKEPEQLRKLFI

    5->19

    1801.08

    C81 H133 N21 O25

    7.1

    0

    3

    KEPEQLRKLFIGGLS

    9->23

    1715.02

    C78 H131 N21 O22

    10.02

    1

    4

    QLRKLFIGGLSFETT

    13->27

    1710.01

    C79 H128 N20 O22

    10.27

    1

    5

    LFIGGLSFETTDESL

    17->31

    1628.81

    C74 H113 N15 O26

    2.87

    -3

    6

    GLSFETTDESLRSHF

    21->35

    1725.85

    C75 H112 N20 O27

    4.4

    -1

    7

    ETTDESLRSHFEQWG

    25->39

    1821.89

    C78 H112 N22 O29

    4.04

    -2

    8

    ESLRSHFEQWGTLTD

    29->43

    1805.93

    C79 H116 N22 O27

    4.4

    -1

    9

    SHFEQWGTLTDCVVM

    33->47

    1752.98

    C77 H113 N19 O24 S2

    3.98

    -1

    10

    QWGTLTDCVVMRDPN

    37->51

    1734.96

    C73 H115 N21 O24 S2

    3.88

    -1

    11

    LTDCVVMRDPNTKRS

    41->55

    1735.01

    C70 H123 N23 O24 S2

    8.93

    1

    12

    VVMRDPNTKRSRGFG

    45->59

    1719.98

    C72 H122 N26 O21 S

    12.19

    3

    13

    DPNTKRSRGFGFVTY

    49->63

    1744.93

    C78 H117 N23 O23

    10.56

    2

    14

    KRSRGFGFVTYATVE

    53->67

    1717.95

    C78 H120 N22 O22

    10.56

    2

    15

    GFGFVTYATVEEVDA

    57->71

    1604.74

    C74 H105 N15 O25

    2.87

    -3

    16

    VTYATVEEVDAAMNA

    61->75

    1583.74

    C67 H106 N16 O26 S

    2.87

    -3

    17

    TVEEVDAAMNARPHK

    65->79

    1667.86

    C69 H114 N22 O24 S

    5.34

    0

    18

    VDAAMNARPHKVDGR

    69->83

    1636.84

    C67 H113 N25 O21 S

    10.26

    2

    19

    MNARPHKVDGRVVEP

    73->87

    1704.96

    C72 H121 N25 O21 S

    10.26

    2

    20

    PHKVDGRVVEPKRAV

    77->91

    1686.96

    C74 H127 N25 O20

    10.69

    3

    21

    DGRVVEPKRAVSRED

    81->95

    1712.88

    C70 H121 N25 O25

    7.16

    0

    22

    VEPKRAVSREDSQRP

    85->99

    1753.94

    C72 H124 N26 O25

    10.24

    1

    23

    RAVSREDSQRPGAHL

    89->103

    1678.83

    C68 H115 N27 O23

    10.98

    2

    24

    REDSQRPGAHLTVKK

    93->107

    1721.93

    C72 H124 N26 O23

    10.69

    3

    25

    QRPGAHLTVKKIFVG

    97->111

    1650.97

    C76 H127 N23 O18

    11.77

    4

    26

    AHLTVKKIFVGGIKE

    101->115

    1639.98

    C77 H130 N20 O19

    10.41

    3

    27

    VKKIFVGGIKEDTEE

    105->119

    1691.93

    C76 H126 N18 O25

    4.64

    -1

    28

    FVGGIKEDTEEHHLR

    109->123

    1766.93

    C77 H119 N23 O25

    5.23

    0

    29

    IKEDTEEHHLRDYFE

    113->127

    1961.09

    C86 H125 N23 O30

    4.39

    -2

    30

    TEEHHLRDYFEQYGK

    117->131

    1952.08

    C87 H122 N24 O28

    5.23

    0

    31

    HLRDYFEQYGKIEVI

    121->135

    1910.16

    C89 H132 N22 O25

    5.34

    0

    32

    YFEQYGKIEVIEIMT

    125->139

    1863.17

    C87 H131 N17 O26 S

    3.73

    -2

    33

    YGKIEVIEIMTDRGS

    129->143

    1710.97

    C74 H123 N19 O25 S

    4.43

    -1

    34

    EVIEIMTDRGSGKKR

    133->147

    1718.99

    C71 H127 N23 O24 S

    10.01

    1

    35

    IMTDRGSGKKRGFAF

    137->151

    1670.95

    C73 H119 N23 O20 S

    11.56

    3

    36

    RGSGKKRGFAFVTFD

    141->155

    1672.9

    C76 H117 N23 O20

    11.56

    3

    37

    KKRGFAFVTFDDHDS

    145->159

    1769.93

    C80 H116 N22 O24

    7.97

    1

    38

    FAFVTFDDHDSVDKI

    149->163

    1755.9

    C81 H114 N18 O26

    3.85

    -2

    39

    TFDDHDSVDKIVIQK

    153->167

    1759.92

    C77 H122 N20 O27

    4.47

    -1

    40

    HDSVDKIVIQKYHTV

    157->171

    1782.01

    C80 H128 N22 O24

    8

    2

    41

    DKIVIQKYHTVNGHN

    161->175

    1765.96

    C78 H124 N24 O23

    9.64

    3

    42

    IQKYHTVNGHNCEVR

    165->179

    1797.99

    C76 H120 N26 O23 S

    8.82

    3

    43

    HTVNGHNCEVRKALS

    169->183

    1664.84

    C68 H113 N25 O22 S

    8.94

    3

    44

    GHNCEVRKALSKQEM

    173->187

    1729.98

    C70 H120 N24 O23 S2

    8.89

    2

    45

    EVRKALSKQEMASAS

    177->191

    1634.87

    C67 H119 N21 O24 S

    10.02

    1

    46

    ALSKQEMASASSSQR

    181->195

    1580.74

    C62 H109 N21 O25 S

    10.27

    1

    47

    QEMASASSSQRGR

    185->197

    1394.49

    C52 H91 N21 O22 S

    11.04

    1

    Peptide Count:  47

             

    B: Heterogeneous nuclear ribonucleoprotein A1 isoform a [Homo sapiens]

    Sequence in FASTA format

    >gi|4504445|ref|NP_002127.1| heterogeneous nuclear ribonucleoprotein A1 isoform a [Homo sapiens]
    MSKSESPKEPEQLRKLFIGGLSFETTDESLRSHFEQWGTLTDCVVMRDPNTKRSRGFGFVTYATVEEVDAAMNARPHKVD
    GRVVEPKRAVSREDSQRPGAHLTVKKIFVGGIKEDTEEHHLRDYFEQYGKIEVIEIMTDRGSGKKRGFAFVTFDDHDSVD
    KIVIQKYHTVNGHNCEVRKALSKQEMASASSSQRGRSGSGNFGGGRGGGFGGNDNFGRGGNFSGRGGFGGSRGGGGYGGS
    GDGYNGFGNDGSNFGGGGSYNDFGNYNNQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGYGGSSSSSSYGSGRRF

    Peptide library of 15mers with an overlap of 11 amino acids for hnRP A1 a, human

    #

    Peptide

    Position

    Mw

    Chemical Formula

    pI

    Charge

    1

    GGSKSESPKEPEQLR

    1->15

    1628.76

    C67 H113 N21 O26

    7.1

    0

    2

    SESPKEPEQLRKLFI

    5->19

    1801.08

    C81 H133 N21 O25

    7.1

    0

    3

    KEPEQLRKLFIGGLS

    9->23

    1715.02

    C78 H131 N21 O22

    10.02

    1

    4

    QLRKLFIGGLSFETT

    13->27

    1710.01

    C79 H128 N20 O22

    10.27

    1

    5

    LFIGGLSFETTDESL

    17->31

    1628.81

    C74 H113 N15 O26

    2.87

    -3

    6

    GLSFETTDESLRSHF

    21->35

    1725.85

    C75 H112 N20 O27

    4.4

    -1

    7

    ETTDESLRSHFEQWG

    25->39

    1821.89

    C78 H112 N22 O29

    4.04

    -2

    8

    ESLRSHFEQWGTLTD

    29->43

    1805.93

    C79 H116 N22 O27

    4.4

    -1

    9

    SHFEQWGTLTDCVVM

    33->47

    1752.98

    C77 H113 N19 O24 S2

    3.98

    -1

    10

    QWGTLTDCVVMRDPN

    37->51

    1734.96

    C73 H115 N21 O24 S2

    3.88

    -1

    11

    LTDCVVMRDPNTKRS

    41->55

    1735.01

    C70 H123 N23 O24 S2

    8.93

    1

    12

    VVMRDPNTKRSRGFG

    45->59

    1719.98

    C72 H122 N26 O21 S

    12.19

    3

    13

    DPNTKRSRGFGFVTY

    49->63

    1744.93

    C78 H117 N23 O23

    10.56

    2

    14

    KRSRGFGFVTYATVE

    53->67

    1717.95

    C78 H120 N22 O22

    10.56

    2

    15

    GFGFVTYATVEEVDA

    57->71

    1604.74

    C74 H105 N15 O25

    2.87

    -3

    16

    VTYATVEEVDAAMNA

    61->75

    1583.74

    C67 H106 N16 O26 S

    2.87

    -3

    17

    TVEEVDAAMNARPHK

    65->79

    1667.86

    C69 H114 N22 O24 S

    5.34

    0

    18

    VDAAMNARPHKVDGR

    69->83

    1636.84

    C67 H113 N25 O21 S

    10.26

    2

    19

    MNARPHKVDGRVVEP

    73->87

    1704.96

    C72 H121 N25 O21 S

    10.26

    2

    20

    PHKVDGRVVEPKRAV

    77->91

    1686.96

    C74 H127 N25 O20

    10.69

    3

    21

    DGRVVEPKRAVSRED

    81->95

    1712.88

    C70 H121 N25 O25

    7.16

    0

    22

    VEPKRAVSREDSQRP

    85->99

    1753.94

    C72 H124 N26 O25

    10.24

    1

    23

    RAVSREDSQRPGAHL

    89->103

    1678.83

    C68 H115 N27 O23

    10.98

    2

    24

    REDSQRPGAHLTVKK

    93->107

    1721.93

    C72 H124 N26 O23

    10.69

    3

    25

    QRPGAHLTVKKIFVG

    97->111

    1650.97

    C76 H127 N23 O18

    11.77

    4

    26

    AHLTVKKIFVGGIKE

    101->115

    1639.98

    C77 H130 N20 O19

    10.41

    3

    27

    VKKIFVGGIKEDTEE

    105->119

    1691.93

    C76 H126 N18 O25

    4.64

    -1

    28

    FVGGIKEDTEEHHLR

    109->123

    1766.93

    C77 H119 N23 O25

    5.23

    0

    29

    IKEDTEEHHLRDYFE

    113->127

    1961.09

    C86 H125 N23 O30

    4.39

    -2

    30

    TEEHHLRDYFEQYGK

    117->131

    1952.08

    C87 H122 N24 O28

    5.23

    0

    31

    HLRDYFEQYGKIEVI

    121->135

    1910.16

    C89 H132 N22 O25

    5.34

    0

    32

    YFEQYGKIEVIEIMT

    125->139

    1863.17

    C87 H131 N17 O26 S

    3.73

    -2

    33

    YGKIEVIEIMTDRGS

    129->143

    1710.97

    C74 H123 N19 O25 S

    4.43

    -1

    34

    EVIEIMTDRGSGKKR

    133->147

    1718.99

    C71 H127 N23 O24 S

    10.01

    1

    35

    IMTDRGSGKKRGFAF

    137->151

    1670.95

    C73 H119 N23 O20 S

    11.56

    3

    36

    RGSGKKRGFAFVTFD

    141->155

    1672.9

    C76 H117 N23 O20

    11.56

    3

    37

    KKRGFAFVTFDDHDS

    145->159

    1769.93

    C80 H116 N22 O24

    7.97

    1

    38

    FAFVTFDDHDSVDKI

    149->163

    1755.9

    C81 H114 N18 O26

    3.85

    -2

    39

    TFDDHDSVDKIVIQK

    153->167

    1759.92

    C77 H122 N20 O27

    4.47

    -1

    40

    HDSVDKIVIQKYHTV

    157->171

    1782.01

    C80 H128 N22 O24

    8

    2

    41

    DKIVIQKYHTVNGHN

    161->175

    1765.96

    C78 H124 N24 O23

    9.64

    3

    42

    IQKYHTVNGHNCEVR

    165->179

    1797.99

    C76 H120 N26 O23 S

    8.82

    3

    43

    HTVNGHNCEVRKALS

    169->183

    1664.84

    C68 H113 N25 O22 S

    8.94

    3

    44

    GHNCEVRKALSKQEM

    173->187

    1729.98

    C70 H120 N24 O23 S2

    8.89

    2

    45

    EVRKALSKQEMASAS

    177->191

    1634.87

    C67 H119 N21 O24 S

    10.02

    1

    46

    ALSKQEMASASSSQR

    181->195

    1580.74

    C62 H109 N21 O25 S

    10.27

    1

    47

    QEMASASSSQRGR

    185->197

    1394.49

    C52 H91 N21 O22 S

    11.04

    1

    Peptide Count:  47

             

    C: Heterogeneous nuclear ribonucleoprotein A1 isoform b [Homo sapiens] {hnRNP A1 b, human}

    Sequence in FASTA format

    >gi|14043070|ref|NP_112420.1| heterogeneous nuclear ribonucleoprotein A1 isoform b [Homo sapiens]

    MSKSESPKEPEQLRKLFIGGLSFETTDESLRSHFEQWGTLTDCVVMRDPNTKRSRGFGFVTYATVEEVDAAMNARPHKVD
    GRVVEPKRAVSREDSQRPGAHLTVKKIFVGGIKEDTEEHHLRDYFEQYGKIEVIEIMTDRGSGKKRGFAFVTFDDHDSVD
    KIVIQKYHTVNGHNCEVRKALSKQEMASASSSQRGRSGSGNFGGGRGGGFGGNDNFGRGGNFSGRGGFGGSRGGGGYGGS
    GDGYNGFGNDGGYGGGGPGYSGGSRGYGSGGQGYGNQGSGYGGSGSYDSYNNGGGGGFGGGSGSNFGGGGSYNDFGNYNN
    QSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGYGGSSSSSSYGSGRRF

    Peptide library of 15mers with an overlap of 11 amino acids for hnRNP A1 b, human

    #

    Peptide

    Position

    Mw

    Chemical Formula

    pI

    Charge

    1

    MSKSESPKEPEQLRK

    1->15

    1774.03

    C74 H128 N22 O26 S

    9.8

    1

    2

    ESPKEPEQLRKLFIG

    5->19

    1771.05

    C80 H131 N21 O24

    7.1

    0

    3

    EPEQLRKLFIGGLSF

    9->23

    1734.03

    C81 H128 N20 O22

    7.07

    0

    4

    LRKLFIGGLSFETTD

    13->27

    1696.97

    C78 H125 N19 O23

    7.04

    0

    5

    FIGGLSFETTDESLR

    17->31

    1671.84

    C74 H114 N18 O26

    3.66

    -2

    6

    LSFETTDESLRSHFE

    21->35

    1797.92

    C78 H116 N20 O29

    4.04

    -2

    7

    TTDESLRSHFEQWGT

    25->39

    1793.88

    C77 H112 N22 O28

    4.4

    -1

    8

    SLRSHFEQWGTLTDC

    29->43

    1779.95

    C77 H114 N22 O25 S

    5.19

    0

    9

    HFEQWGTLTDCVVMR

    33->47

    1822.09

    C80 H120 N22 O23 S2

    5.19

    0

    10

    WGTLTDCVVMRDPNT

    37->51

    1707.94

    C72 H114 N20 O24 S2

    3.88

    -1

    11

    TDCVVMRDPNTKRSR

    41->55

    1778.04

    C70 H124 N26 O24 S2

    10.25

    2

    12

    VMRDPNTKRSRGFGF

    45->59

    1768.03

    C76 H122 N26 O21 S

    12.19

    3

    13

    PNTKRSRGFGFVTYA

    49->63

    1700.92

    C77 H117 N23 O21

    11.46

    3

    14

    RSRGFGFVTYATVEE

    53->67

    1718.9

    C77 H115 N21 O24

    7.03

    0

    15

    FGFVTYATVEEVDAA

    57->71

    1618.77

    C75 H107 N15 O25

    2.87

    -3

    16

    TYATVEEVDAAMNAR

    61->75

    1640.8

    C68 H109 N19 O26 S

    3.66

    -2

    17

    VEEVDAAMNARPHKV

    65->79

    1665.88

    C70 H116 N22 O23 S

    5.34

    0

    18

    DAAMNARPHKVDGRV

    69->83

    1636.84

    C67 H113 N25 O21 S

    10.26

    2

    19

    NARPHKVDGRVVEPK

    73->87

    1701.93

    C73 H124 N26 O21

    10.69

    3

    20

    HKVDGRVVEPKRAVS

    77->91

    1676.92

    C72 H125 N25 O21

    10.69

    3

    21

    GRVVEPKRAVSREDS

    81->95

    1684.87

    C69 H121 N25 O24

    10.24

    1

    22

    EPKRAVSREDSQRPG

    85->99

    1711.86

    C69 H118 N26 O25

    10.24

    1

    23

    AVSREDSQRPGAHLT

    89->103

    1623.75

    C66 H110 N24 O24

    8.02

    1

    24

    EDSQRPGAHLTVKKI

    93->107

    1678.9

    C72 H123 N23 O23

    10.02

    2

    25

    RPGAHLTVKKIFVGG

    97->111

    1579.89

    C73 H122 N22 O17

    11.77

    4

    26

    HLTVKKIFVGGIKED

    101->115

    1683.99

    C78 H130 N20 O21

    9.8

    2

    27

    KKIFVGGIKEDTEEH

    105->119

    1729.94

    C77 H124 N20 O25

    5.44

    0

    28

    VGGIKEDTEEHHLRD

    109->123

    1734.84

    C72 H115 N23 O27

    4.68

    -1

    29

    KEDTEEHHLRDYFEQ

    113->127

    1976.06

    C85 H122 N24 O31

    4.39

    -2

    30

    EEHHLRDYFEQYGKI

    117->131

    1964.13

    C89 H126 N24 O27

    5.23

    0

    31

    LRDYFEQYGKIEVIE

    121->135

    1902.14

    C88 H132 N20 O27

    4.04

    -2

    32

    FEQYGKIEVIEIMTD

    125->139

    1815.08

    C82 H127 N17 O27 S

    3.47

    -3

    33

    GKIEVIEIMTDRGSG

    129->143

    1604.84

    C67 H117 N19 O24 S

    4.43

    -1

    34

    VIEIMTDRGSGKKRG

    133->147

    1646.92

    C68 H123 N23 O22 S

    10.69

    2

    35

    MTDRGSGKKRGFAFV

    137->151

    1656.92

    C72 H117 N23 O20 S

    11.56

    3

    36

    GSGKKRGFAFVTFDD

    141->155

    1631.8

    C74 H110 N20 O22

    10.02

    1

    37

    KRGFAFVTFDDHDSV

    145->159

    1740.89

    C79 H113 N21 O24

    5.24

    0

    38

    AFVTFDDHDSVDKIV

    149->163

    1707.85

    C77 H114 N18 O26

    3.85

    -2

    39

    FDDHDSVDKIVIQKY

    153->167

    1821.99

    C82 H124 N20 O27

    4.47

    -1

    40

    DSVDKIVIQKYHTVN

    157->171

    1758.97

    C78 H127 N21 O25

    7.9

    1

    41

    KIVIQKYHTVNGHNC

    161->175

    1754.01

    C77 H124 N24 O21 S

    9.67

    4

    42

    QKYHTVNGHNCEVRK

    165->179

    1813

    C76 H121 N27 O23 S

    9.67

    4

    43

    TVNGHNCEVRKALSK

    169->183

    1655.87

    C68 H118 N24 O22 S

    10.02

    3

    44

    HNCEVRKALSKQEMA

    173->187

    1744.01

    C71 H122 N24 O23 S2

    8.89

    2

    45

    VRKALSKQEMASASS

    177->191

    1592.83

    C65 H117 N21 O23 S

    10.7

    2

    46

    LSKQEMASASSSQRG

    181->195

    1566.71

    C61 H107 N21 O25 S

    10.27

    1

    47

    EMASASSSQRGRSGS

    185->199

    1497.57

    C55 H96 N22 O25 S

    11.04

    1

    48

    ASSSQRGRSGSGNFG

    189->203

    1454.47

    C56 H91 N23 O23

    12.49

    2

    49

    QRGRSGSGNFGGGRG

    193->207

    1449.49

    C56 H92 N26 O20

    12.81

    3

    50

    SGSGNFGGGRGGGFG

    197->211

    1270.26

    C52 H75 N19 O19

    11.18

    1

    51

    NFGGGRGGGFGGNDN

    201->215

    1382.34

    C56 H79 N21 O21

    6.95

    0

    52

    GRGGGFGGNDNFGRG

    205->219

    1424.43

    C58 H85 N23 O20

    11.04

    1

    53

    GFGGNDNFGRGGNFS

    209->223

    1502.5

    C64 H87 N21 O22

    6.95

    0

    54

    NDNFGRGGNFSGRGG

    213->227

    1511.51

    C61 H90 N24 O22

    11.04

    1

    55

    GRGGNFSGRGGFGGS

    217->231

    1369.4

    C56 H84 N22 O19

    12.49

    2

    56

    NFSGRGGFGGSRGGG

    221->235

    1369.4

    C56 H84 N22 O19

    12.49

    2

    57

    RGGFGGSRGGGGYGG

    225->239

    1298.32

    C53 H79 N21 O18

    11.21

    2

    58

    GGSRGGGGYGGSGDG

    229->243

    1197.12

    C45 H68 N18 O21

    6.86

    0

    59

    GGGGYGGSGDGYNGF

    233->247

    1321.26

    C56 H72 N16 O22

    3.1

    -1

    60

    YGGSGDGYNGFGNDG

    237->251

    1436.35

    C60 H77 N17 O25

    2.92

    -2

    61

    GDGYNGFGNDGGYGG

    241->255

    1406.32

    C59 H75 N17 O24

    2.92

    -2

    62

    NGFGNDGGYGGGGPG

    245->259

    1282.22

    C53 H71 N17 O21

    3.1

    -1

    63

    NDGGYGGGGPGYSGG

    249->263

    1271.2

    C52 H70 N16 O22

    3.1

    -1

    64

    YGGGGPGYSGGSRGY

    253->267

    1391.41

    C60 H82 N18 O21

    9.43

    1

    65

    GPGYSGGSRGYGSGG

    257->271

    1315.31

    C54 H78 N18 O21

    9.63

    1

    66

    SGGSRGYGSGGQGYG

    261->275

    1346.32

    C54 H79 N19 O22

    9.63

    1

    67

    RGYGSGGQGYGNQGS

    265->279

    1444.42

    C58 H85 N21 O23

    9.63

    1

    68

    SGGQGYGNQGSGYGG

    269->283

    1345.28

    C54 H76 N18 O23

    5.96

    0

    69

    GYGNQGSGYGGSGSY

    273->287

    1410.36

    C59 H79 N17 O24

    5.96

    0

    70

    QGSGYGGSGSYDSYN

    277->291

    1498.43

    C62 H83 N17 O27

    3.1

    -1

    71

    YGGSGSYDSYNNGGG

    281->295

    1454.37

    C60 H79 N17 O26

    3.1

    -1

    72

    GSYDSYNNGGGGGFG

    285->299

    1408.34

    C59 H77 N17 O24

    3.1

    -1

    73

    SYNNGGGGGFGGGSG

    289->303

    1244.17

    C50 H69 N17 O21

    6

    0

    74

    GGGGGFGGGSGSNFG

    293->307

    1171.12

    C48 H66 N16 O19

    6.09

    0

    75

    GFGGGSGSNFGGGGS

    297->311

    1201.15

    C49 H68 N16 O20

    6.09

    0

    76

    GSGSNFGGGGSYNDF

    301->315

    1422.37

    C60 H79 N17 O24

    3.1

    -1

    77

    NFGGGGSYNDFGNYN

    305->319

    1582.54

    C69 H87 N19 O25

    3.1

    -1

    78

    GGSYNDFGNYNNQSS

    309->323

    1623.55

    C67 H90 N20 O28

    3.1

    -1

    79

    NDFGNYNNQSSNFGP

    313->327

    1674.64

    C71 H95 N21 O27

    3.1

    -1

    80

    NYNNQSSNFGPMKGG

    317->331

    1614.69

    C67 H99 N21 O24 S

    9.8

    1

    81

    QSSNFGPMKGGNFGG

    321->335

    1484.59

    C63 H93 N19 O21 S

    10.28

    1

    82

    FGPMKGGNFGGRSSG

    325->339

    1455.6

    C62 H94 N20 O19 S

    11.6

    2

    83

    KGGNFGGRSSGPYGG

    329->343

    1397.45

    C59 H88 N20 O20

    10.58

    2

    84

    FGGRSSGPYGGGGQY

    333->347

    1446.49

    C63 H87 N19 O21

    9.63

    1

    85

    SSGPYGGGGQYFAKP

    337->351

    1472.57

    C67 H93 N17 O21

    9.52

    1

    86

    YGGGGQYFAKPRNQG

    341->355

    1599.71

    C71 H102 N22 O21

    10.19

    2

    87

    GQYFAKPRNQGGYGG

    345->359

    1599.71

    C71 H102 N22 O21

    10.19

    2

    88

    AKPRNQGGYGGSSSS

    349->363

    1452.49

    C58 H93 N21 O23

    10.58

    2

    89

    NQGGYGGSSSSSSYG

    353->367

    1394.32

    C55 H79 N17 O26

    5.96

    0

    90

    YGGSSSSSSYGSGRR

    357->371

    1494.5

    C59 H91 N21 O25

    10.42

    2

    91

    SSSSSYGSGRRF

    361->372

    1277.32

    C52 H80 N18 O20

    11.21

    2

    Peptide Count:  91

     

     

    References

    Barraud P, Allain FH; Solution structure of the two RNA recognition motifs of hnrnp a1 using segmental isotope labeling: how the relative orientation between rrms influences the nucleic acid binding topology.J.Biomol.Nmr (2013) 55 p.119.

    Carolyn J. Decker and Roy Parker; P-Bodies and Stress Granules: Possible Roles in the Control of Translation and mRNA Degradation. Cold Spring
    Harb Perspect Biol a012286 First published online July 3, 2012.


    Frank Desiere, Eric W. Deutsch, Alexey I. Nesvizhskii, Parag Mallick, Nichole King, Jimmy K. Eng, Alan Aderem, Rose Boyle, Erich Brunner, Samuel Donohoe, Nelson Fausto, Ernst Hafen, Lee Hood, Michael G. Katze, Kathleen Kennedy, Floyd Kregenow, Hookeun Lee, Biaoyang Lin, Dan Martin, Jeff Ranish, David J. Rawlings, Lawrence E. Samelson, Yuzuru Shiio, Julian Watts, Bernd Wollscheid, Michael E. Wright, Wei Yan, Lihong Yang, Eugene Yi, Hui Zhang and Ruedi Aebersold Genome Biology 2004, 6:R9
    Integration of Peptide Sequences Obtained by High-Throughput Mass Spectrometry with the Human Genome.

    Eric W Deutsch, Henry Lam & Ruedi Aebersold EMBO reports 9, 5, 429–434 (2008) PeptideAtlas: a resource for target selection for emerging targeted proteomics workflows

    Domon B, Aebersold R. Science. 2006 Apr 14;312(5771):212-7 Mass spectrometry and protein analysis.

    Lange V, Malmström JA, Didion J, King NL, Johansson BP, Schäfer J, Rameseder J, Wong CH, Deutsch EW, Brusniak MY, Bühlmann P, Björck L, Domon B, Aebersold R. Mol Cell Proteomics. 2008 Apr 13. Targeted quantitative analysis of Streptococcus pyogenes virulence factors by multiple reaction monitoring.

    Paola Picotti, Mathieu Clément-Ziza, Henry Lam, David S. Campbell, Alexander Schmidt, Eric W. Deutsch, Hannes Röst, Zhi Sun, Oliver Rinner, Lukas Reiter, Qin Shen, Jacob J. Michaelson, Andreas Frei, Simon Alberti, Ulrike Kusebauch, Bernd Wollscheid, Robert L. Moritz, Andreas Beyer & Ruedi Aebersold Nature. 2013 Feb 14;494(7436):266-70. doi: 10.1038/nature11835. Epub 2013 Jan 20. A complete mass-spectrometric map of the yeast proteome applied to quantitative trait analysis

    Paola Picotti, Henry Lam, David Campbell, Eric W. Deutsch, Hamid Mirzaei, Jeff Ranish, Bruno Domon and Ruedi Aebersold Nature Methods A database of mass spectrometric assays for the yeast proteome. Nat Methods. 2008 November; 5(11): 913–914.

    B L Robberson, G J Cote, and S M Berget; Exon definition may facilitate splice site selection in RNAs with multiple exons. Mol Cell Biol. 1990 January; 10(1): 84–94. PMCID: PMC360715

    Other resources

    HNRNPA1: provided by HGNC (http://www.genenames.org/), heterogeneous nuclear ribonucleoprotein A1: provided by HGNC. Primary source: HGNC:5031, See related: Ensembl:ENSG00000135486; HPRD:01242; MIM:164017; Vega:OTTHUMG00000169702; Gene type: protein coding; Organism: Homo sapiens. Lineage: Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Primates; Haplorrhini; Catarrhini; Hominidae; Homo. Also known as: HNRPA1; HNRPA1L3; hnRNP A1; hnRNP-A1

    0 0

    BNAs for Duplex and Triplex Formation

    2’,4’-BNA (LNA) with a five-membered bridged structure is insufficiently resistant to nucleases, and fully modified 2’,4’-BNA (LNA) oligonucleotides do not have the flexibility required for efficient triplex formation.

    BNA analogues with increased steric bulk and less conformational restriction were developed. 2’,4’-BNACOC has a seven membered bridged structure and exhibits dramatically improved nuclease resistance.

    NA-5 = BNACOC: This modification conversely affects duplex stability [i.e., duplexes formed with this nucleic acid analogue are less stable than those formed by 2’,4’-BNA (LNA)].

    ENA, with a six-membered bridged structure, has slightly lower duplex-forming ability and significantly higher nuclease resistance than 2’,4’-BNA (LNA).

    Triplex formation with ENA provided variable results compared to that of 2’,4’-BNA (LNA).

    2’,4’-BNANC, has a six-membered bridged structure with a unique structural feature (N-O bond) in the sugar moiety

    The bridged moiety was designed to have a nitrogen atom, which can:

    1. Act as a conjugation site
    2. Improve duplex and triplex stability by lowering repulsions between the negatively charged backbone phosphates

     

    The nitrogen atom on the bridge can be functionalized by hydrophobic and hydrophilic groups, by steric bulk, or by appropriate functional moieties to:

    1. (Control affinity toward complementary strands
    2. Regulate resistance against nuclease degradation, and
    3. Allows synthesis of functional molecules designed for specific applications in genomics.
    In addition to the above possibilities, the N-O bond could be cleaved under appropriate reductive conditions to modulate the hybridizing properties of 2’,4’-BNANC.

     

    I. Duplex formation with RNA based on Tm

    Target ssRNA: 5’-r(AGCAAAAAACGC)-3’

     

    Duplex: 5’-r(AGCAAAAAACGC)-3’
    3’-d(TCGTTTTTTGCG)-5’

    Duplex formation with 2’,4’-BNANC[NH] chimer:

      2’,4’-BNANC[NH] Tm ΔTm ΔTm/BNA
    d Oligo GCGTTTTTTGCT 45    
    1 BNA GCGTTTTTTGCT 51 +6 +6
    3 BNAs GCGTTTTTTGCT 64 +19 +6.3
    3 BNAs GCGTTTTTTGCT 61 +16 +5.3
    6 BNAs GCGTTTTTTGCT 83 +38 +6.3

    Duplex formation with 2’,4’-BNANC[NH] chimer:

      2’,4’-BNANC[NH] Tm ΔTm ΔTm/BNA
    d Oligo GCGTTTTTTGCT 45    
    1 BNA GCGTTTTTTGCT 50 +5 +5
    3 BNAs GCGTTTTTTGCT 63 +18 +6
    3 BNAs GCGTTTTTTGCT 59 +14 +4.7
    6 BNAs GCGTTTTTTGCT 80 +35 +5.8

    Duplex formation with 2’,4’-BNA (LNA) chimer:

      2’,4’-BNANC[NH] Tm ΔTm ΔTm/BNA
    d Oligo GCGTTTTTTGCT 45    
    1 BNA GCGTTTTTTGCT 52 +7 +7
    3 BNAs GCGTTTTTTGCT 62 +17 +5.7
    3 BNAs GCGTTTTTTGCT 60 +15 +5
    6 BNAs GCGTTTTTTGCT 80 +35 +5.8
    Note 1: A dOligo with one LNA monomer binds the strongest.
    Note 2: A dOligo with multiple 2’,4’-BNANC[NMe] monomers bind with higher affinities to RNA.
    Note 3: A dOligo with multiple 2’,4’-BNANC[NMe] monomers bind with similar affinities to RNA when compared to LNA.
    Conclusion: 2’,4’-BNANC[NH] is more specific to RNA. LNA and 2’,4’-BNANC[NMe] are similar.

    II. Duplex formation with DNA based on Tm

    Target ssDNA: 5’-d(AGCAAAAAACGC)-3’

     

    Duplex: 5’-d(AGCAAAAAACGC)-3’
    3’-d(TCGTTTTTTGCG)-5’

    Duplex formation with 2’,4’-BNANC[NH] chimer:

      2’,4’-BNANC[NH] Tm ΔTm ΔTm/BNA
    d Oligo GCGTTTTTTGCT 50    
    1 BNA GCGTTTTTTGCT 51 +1 +1
    3 BNAs GCGTTTTTTGCT 55 +18 +1.7
    3 BNAs GCGTTTTTTGCT 57 +14 +2.3
    6 BNAs GCGTTTTTTGCT 73 +23 +3.8

    Duplex formation with 2’,4’-BNANC[NMe] chimer:

      2’,4’-BNANC[NH] Tm ΔTm ΔTm/BNA
    d Oligo GCGTTTTTTGCT 50    
    1 BNA GCGTTTTTTGCT 49 -1 -1
    3 BNAs GCGTTTTTTTGCT 51 +1 +0.3
    3 BNAs GCGTTTTTTGCT 50 0 0
    6 BNAs GCGTTTTTTGGCT 61 +11 +1.8

    Duplex formation with 2’,4’-BNA(LNA) chimer:

      2’,4’-BNANC[NH] Tm ΔTm ΔTm/BNA
    d Oligo GCGTTTTTTGCT 50    
    1 BNA GCGTTTTTTGCT 53 +3 +3
    3 BNAs GCGTTTTTTTGCT 56 +6 +2
    3 BNAs GCGTTTTTTGCT 54 +4 1.3
    6 BNAs GCGTTTTTTGGCT 67 +17 +2.8
    Note 1: A dOligo with one LNA monomer binds the strongest.
    Note 2: dOligos with multiple 2’,4’-BNANC[NH] monomers bind with higher affinities to DNA than dOligos with 2’,4’-BNANC[NMe].
    Note 3: A dOligo with multiple 2’,4’-BNANC[NMe] monomers bind with higher affinities to DNA when compared to LNA oligos.
    Conclusion: Oligos with several 2’,4’-BNANC[NMe] bind more strongly to DNA when compared to oligos with 2’,4’-BNANC[NMe] or LNA monomers.

    Tm Values of Duplexes

    Formed by 2’,4’-BNANC- and 2’,4’-BNA (LNA)-Modified Oligonucleotides with Complementary ssRNA Containing a Single-Mismatch Basea,b
    Tm (ΔTm= Tm (mismatch) -Tm (match)(oC)
    T (oligonucleotide) X ) A (match) U G C
    GCGTTTTTTGCT 45 33(-12) 43(-3) 30(-15)
    2',4'-BNANC[NH]
    GCGTTTTTTGCT
    51 37 (-14) 46(-5) 34 (-17)
    2',4'-BNANC[NMe]
    GCGTTTTTTGCT
    50 37 (-13) 48 (-2) 34 (-16)
    2',4'-BNA (LNA)
    GCGTTTTTTGCT
    52 39 (-13) 47 (-5) 35 (-17)
    a Modified oligonucleotides, 5'-d(GCGTTTTTTGCT)-3'; Target ssRNA strand, 3'-(CGCAAXAAACGA)-5'. b Conditions: 10 mM sodium phosphate buffer (pH 7.2) containing 100 mM NaCl; strand concentration) 4 µM.

    Conclusion:

    ΔTmvalues in the Table show that any mismatched base in the target RNA strand resulted in a substantial decrease in the Tm of duplexes formed with 2’,4’-BNANC-modified oligonucleotides.

    ΔTmvalues of duplexes formed with 2’,4’-BNANC[NH]-modified oligonucleotide having T-U, T-G, and T-C arrangements are -14, -5, -17 °C.

    These are lower than those of the corresponding natural DNA-RNA duplexes (ΔTm= -12, -3, and -15 °C, respectively).

    Similar to those exhibited by duplexes formed with the 2’,4’-BNA (LNA) oligonucleotide (ΔTm= -13, -5, and -17 °C, respectively).

    The mismatch discrimination profiles of 2’,4’-BNANC[NMe] and [NBn] were also similar to that of 2’,4’-BNA (LNA), except in the case of the T-G arrangement for the 2’,4’-BNANC[NMe] derivative, as shown in the Table.

    Thus, it appears that 2’,4’-BNANC not only exhibits high-affinity RNA selective hybridization, but is also highly selective in recognizing bases.

    III. Triplex Formation and Triplex Stability.

    Applications in antigene and gene repair technologies require the formation of stable triplexes at physiological pH.
    Rahman et al in 2007 showed that 2’,4’-BNANC[NH] has triplex-forming characteristics when compared to those of 2’,4’-BNA (LNA) and ENA.
    Details of triplex formation by three 2’,4’-BNANC analogues (2’,4’-BNANC[NH], [NMe], and [NBn]) in the absence and presence of divalent metal (Mg2+) are described here.
    triplex-forming ability of the 2’,4’-BNANC analogues against a 21 bp double-stranded DNA (dsDNA) in the absence of divalent metal is summarized in next Table.
    A single modification of the triplex-forming oligonucleotide (TFO) with 2’,4’-BNANC[NH] (TFO) increased the Tmof the triplex by 11 °C, which is equal to that exhibited by the triplex of 2’,4’-BNA (LNA)-modified TFO.

    The Tm of the triplex formed by the corresponding 2’,4’-BNANC-[NMe]-TFO is 5 °C higher than that of natural-TFO, and that of 2’,4’-BNANC[NBn]-TFO is equal to that of the natural oligonucleotide. In the case of 2’,4’-BNANC[NH], increasing the number of modifications (TFOs) greatly enhanced triplex thermal stability (ΔTm/mod. = +6.2 to +9 °C), which is similar to or even higher than that of the corresponding 2’,4’-BNA (LNA)-TFOs (ΔTm/mod. = +4.9 to +8.7 °C).

    The thermal stability of the triplex formed by the corresponding 2’,4’-BNANC[NMe]-TFOs, although significantly higher than that obtained with natural DNA-TFO, is lower than that of 2’,4’-BNANC[NH]- and 2’,4’-BNA (LNA)-TFOs.

    Modification with 2’,4’-BNANC-[NBn] units did not affect the thermal stability of the triplex; that is, the thermal stability was similar to that of natural DNA-TFO. The lower stability of the 2’,4’-BNANC-[NMe] and [NBn] triplexes might be due to unfavorable steric interaction with dsDNA, induced by the methyl or benzyl groups.
    (Rahman, S. M. A.; Seki, S.; Obika, S.; Haitani, S.; Miyashita, K.; Imanishi, T. Angew. Chem., Int. Ed. 2007, 46, 4306{c}).

    Tm Values of Triplexes Formed by 2’,4’-BNANC- and 2’,4’-BNA (LNA)-Modified Oligonucleotides with Complementary dsDNA in the Absence of Mg2+.a,b

    No Mg2+ are present!


    TmTm/mod.) (°C)

    oligonucleotides T= 2’,4’-BNANC 2’,4’-BNA (LNA)

    [NH] [NMe] [NBn]
    d(TTTTTmCTTTmCTmCTmCT) 33 33 33 33
    d(TTTTTmCTTTmCTmCTmCT 44 (+11.0)c 38 (+5.0) 33 (+0.0) 44 (+11.0)c
    d(TTTTTmCTTTmCTmCTmCT) 60 (+9.0) c 47 (+4.6) 32 (-0.3) 59 (+8.7) c
    d(TTTTTmCTTTmCTmCTmCT) 59 (+8.7) c 42 (+3.0) 31 (-0.7) 52 (+6.3) c
    d(TTTTTmCTTTmCTmCTmCT 58 (+6.3) c 45 (+3.0)   57 (+6.0) c
    d(TTTTTmCTTTmCTmCTmCT) 64 (+6.2) c 50 (+3.4)   65 (+6.4) c
    d(TTTTTmCTTTmCTmCTmCT) 78 (+6.4) c 59 (+3.7)   67 (+4.9) c
    d(TTTTTmCTTTmCTmCTmCT) 80 (+3.1) c <5 (<-2)   <5 (<-2) c
    a Target dsDNA: 5’-d(GCTAAAAAGAAAGAGAGATCG)-3’
    3’-d(CGATTTTTCTTTCTCTCTAGC)-5’
    underlined portion indicates the target site for triplex formation. b Conditions: 7 mM sodium phosphate buffer (pH 7.0) containing 140 mM KCl; strand concentration = 1.5 µM. c Data from Rahman et al. 2007.

    Conclusion:
    2’,4’-BNANC[NH] form more stable TFOs when compared to 2’,4’-BNANC[NMe] and LNAs.

    Proper spacing of the monomers appears to make the binding stronger.

    Best placement of BNANC[NH] monomers if no Mg2+ ions are present for triplex probes are:

    d(TTTTTmCTTTmCTmCTmCT)
    d(TTTTTmCTTTmCTmCTmCT)

    TmValues of Triplexes Formed by 2’,4’-BNANC- and 2’,4’-BNA (LNA)-Modified Oligonucleotides with Complementary dsDNA in the Presence of Mg2+. a,b

    10 mM Mg2+ are present!


    Tm(ΔTm/mod.) (°C)

    oligonucleotides T = 2’,4’-BNANC 2’,4’-BNA (LNA)

    [NH] [NMe] [NBn]
    d(TTTTTmCTTTmCTmCTmCT) 43 43 43 43
    d(TTTTTmCTTTmCTmCTmCT 55 (+12.0) 49 (+6.0) 44 (+1.0) 55 (+12.0)
    d(TTTTTmCTTTmCTmCTmCT) 73 (+10.0) 61 (+6.0) 45 (+0.7) 72 (+ 9.7)
    d(TTTTTmCTTTmCTmCTmCT) 71 (+ 9.3) 54 (+3.6) 44 (+0.3) 64 (+ 7.0)
    a Target dsDNA: 5’-d(GCTAAAAAGAAAGAGAGATCG)-3’
    3’-d(CGATTTTTCTTTCTCTCTAGC)-5’

    underlined portion indicates the target site for triplex formation. b Conditions: 7 mM sodium phosphate buffer (pH 7.0) containing 140 mM KCl; strand concentration = 1.5 µM. c Data from Rahman et al. 2007.

    Conclusion:

    Oligonucleotides with 2’,4’-BNANC[NH] monomers form more stable TFOs when compared to 2’,4’-BNANC[NMe] and LNAs.

    Proper spacing of the monomers appears to make the binding stronger.

    Best placement of BNANC[NH] monomers for triplex probes is:

     

    d(TTTTTmCTTTmCTmCTmCT)

    This design appears to be the best for the design of probes that form stable

    triplexes under physiological conditions!

    This is similar or close to LNA oligos.

    2’,4’-BNANC[NH] oligos form more stable TFOs.

     

    Molecular models of Triplexes

    Molecular modeling of a parallel-motif triplex formed by 2’,4’-BNANC[NH]-TFO with dsDNA. BNA: d(TTTTTmCTTTmCTmCTmCT). (Source: Rahman et al., 2008).

    (A) The overall view of the triplex is depicted.

    The dsDNA is shown as a gray CPK model with the phosphate backbone of the purine strand colored red and purple.

    TFO is shown as a colored tube model with three 20,40-BNANC nitrogens represented in green.

    (B) Expanded view of the area of the triplex containing three consecutive 20,40-BNANC[NH] residues.

    Note that the 20,40-BNANC nitrogens are very close to the phosphate moiety of the purine strand indicating ionic interactions of the compounds.

     


    0 0

    References for BNANC[NH] and [NMe] monomers

    Application BNA Publication Year
    • Hybridization.
    • Nuclease stability
    • Duplex-Formation RNA and DNA

    Rahman SM, Seki S, Utsuki K, Obika S, Miyashita K, Imanishi T.; Synthesis and properties of 2',4'-BNA(NC), a second generation BNA. Nucleic Acids Symp Ser (Oxf). 2005; (49):5-6.

    2005
    • Duplex formation RNA & DNA
    • High RNA Selectivity
    BNANC[NH]
    &
    BNANC[NMe]

    Rahman SM, Seki S, Utsuki K, Obika S, Miyashita K, Imanishi T.; High-affinity RNA mimicking binding of 2',4'-BNANC towards complementary strands: a comparative study with 2',4'-BNA/LNA. 2006 Nucleic Acids Symposium Series No. 50 195–196.

    2006
    • Duplex formation RNA & DNA
    • High RNA Selectivity
    • Thermal Stability
    • Nuclease Resistance
    • TFO
    BNANC[NH]
    &
    BNANC[NMe]

    Rahman SM, Seki S, Utsuki K, Obika S, Miyashita K, Imanishi T.; 2',4'-BNA(NC): a novel bridged nucleic acid analogue with excellent hybridizing and nuclease resistance profiles. Nucleosides Nucleotides Nucleic Acids.2007;26(10-12):1625-8.

    2007
    • Antisense
    • High RNA affinity
    • Better RNA selectivity
    • Higher resistance to nuclease degradation.
    BNANC[NMe]

    Kazuyuki Miyashita, S. M. Abdur Rahman, Sayori Seki, Satoshi Obikaab and Takeshi Imanishi;   N-Methyl substituted 2’,4’-BNANC: a highly nuclease-resistant nucleic acid analogue with high-affinity RNA selective hybridization.  Chem. Commun., 2007, 3765–3767.

    2007
    • dsDNA Sensing Probes
    • ADAC probes
    • Triplex Formation (TFO)
    • Cleavage of phosphoramidate bond
    BNANC[NH]
    &
    BNANC[NMe]

    Satoshi Obika, Masaharu Tomizu, Yoshinori Negoro, Ayako Orita, Osamu Nakagawa, and Takeshi Imanishi;  Double-Stranded DNA-Templated Oligonucleotide Digestion Triggered by Triplex Formation. ChemBioChem 2007, 8, 1924 – 1928. Note: Triplex triggered cleavage of oligonucleotides.

    2007
    • TFO
    • Highly Stable Pyrimidine TFO

    BNANC[NH]

    S. M. Abdur Rahman, Sayori Seki, Satoshi Obika, Sunao Haitani, Kazuyuki Miyashita, and Takeshi Imanishi; Highly Stable Pyrimidine-Motif Triplex Formation at Physiological pH Values by a Bridged Nucleic Acid Analogue. Angew. Chem. Int. Ed. 2007, 46, 4306 –4309.

    2007
    • SELEX
    • DNA Aptamer
    • DNAzymes
    BNANC[NH]

    Masayasu Kuwahara, Satoshi Obika, Jun-ichi Nagashima, Yuki Ohta, Yoshiyuki Suto, Hiroaki Ozaki, Hiroaki Sawai and Takeshi Imanishi; Systematic analysis of enzymatic DNA polymerization using oligo-DNA templates and triphosphate analogs involving 2’,4’-bridged nucleosides. Nucleic Acids Research, 2008, Vol. 36, No. 13 4257–4265.

    2008
    • Sequence Specificity
    • Duplex Formation
    • Triplex Formation (TFO)
     

    Satoshi Obika, S. M. Abdur Rahman, Bingbing Song, Mayumi Onoda, Makoto Koizumi, Koji Morita, Takeshi Imanishi; Synthesis and properties of 3’-amino-2’,4’-BNA, a bridged nucleic acid with a N3’->P5’ phosphoramidate linkage. Bioorganic & Medicinal Chemistry 16 (2008) 9230–9237.

    2008
    • High RNA affinity
    • Better RNA selectivity than LNA
    • Enhanced triplex formation (TFO)
    • Stable triplexes at neutral pH
    • Dramatically improved resistance to nuclease degradation.
    • Antisense
    BNANC[NH]
    &
    BNANC[NMe]

    S. M. Abdur Rahman, Sayori Seki, Satoshi Obika, Haruhisa Yoshikawa, Kazuyuki Miyashita, and Takeshi Imanishi;   Design, Synthesis, and Properties of 2’,4’-BNANC: A Bridged Nucleic Acid Analogue.  J. AM. CHEM. SOC. 2008, 130, 4886-4896.

    BNANC[NMe] has high RNA affinity and better RNA selectivity than LNA. BNANC[NH] enhanced triplex formation ability (TFO)
    Fully modified BNANC[NH] oligonucleotide form stable triplex at neutral pH. BNANC[NH] oligonucleotide dramatically improved resistance to nuclease degradation.

    2008
    • TFO
    BNANC[NH]

    Sasaki K, Rahman SM, Obika S,Imanishi T, Torigoe H Promotion of triplex formation by 2'-O,4'-C-aminomethylene bridged nucleic acid (2',4'-BNA NC) modification. Nucleic acids symposium series (2004) : 52 2008 pg 419-20.

    2008
    • Aptamer capping at 3’-ends
    BNANC[NH

    Yuuya Kasahara, Shunsuke Kitadume, Kunihiko Morihiro, Masayasu Kuwahara, Hiroaki Ozaki, Hiroaki Sawai, Takeshi Imanishi, Satoshi Obika;  Effect of 3’-end capping of aptamer with various 2’,4’-bridged nucleotides: Enzymatic post-modification toward a practical use of polyclonal aptamers. Bioorganic & Medicinal Chemistry Letters 20 (2010) 1626–1629.

    2010
    • siRNA
    BNANC[NH]

    S. M. Abdur Rahman, Hiroyuki Sato, Naoto Tsuda, Sunao Haitani, Keisuke Narukawa, Takeshi Imanishi, Satoshi Obika; RNA interference with 2’,4’-bridged nucleic acid analogues. Bioorganic & Medicinal Chemistry 18 (2010) 3474–3480.
    siRNA to inhibit firefly luciferase expression in CHO-luc cells.

    2010
    • Antisense drug discovery
    • Antisense drug development
    • Antisense medicinal chemistry
    • Antisense in clinical trial
     

    Tsuyoshi Yamamoto, Moeka Nakatani, Keisuke Narukawa & Satoshi Obika;Antisense drug discovery and development Future Med. Chem. (2011) 3(3), 339–365. Overview including BNANCs.

    2011
    • Gene Targeting
    • Duplex Formation
    • Triplex Formation
    • Kinetics
    BNANC[NMe]

    Hidetaka Torigoe and Takeshi ImanishiChemical Modification of Oligonucleotides: A Novel Approach Towards Gene Targeting. InTech. http://dx.doi.org/10.5772/50393

    2012
    • Antisense
    • Inhibition of PCSK9 expression
    • AONs
    BNANC[NMe] PS

    Tsuyoshi Yamamoto, Mariko Harada-Shiba, Moeka Nakatani, Shunsuke Wada, Hidenori Yasuhara, Keisuke Narukawa, Kiyomi Sasaki, Masa-Aki Shibata, Hidetaka Torigoe, Tetsuji Yamaoka, Takeshi Imanishi and Satoshi Obika;  Cholesterol-lowering Action of BNA-based Antisense Oligonucleotides Targeting PCSK9 in Atherogenic Diet-induced Hypercholesterolemic Mice Molecular Therapy–Nucleic Acids (2012) 1

    2012

    Overview of listed papers

    2005:   Rahman SM, Seki S, Utsuki K,Obika S, Miyashita K, Imanishi T.; Synthesis and properties of 2',4'-BNA(NC), a second generation BNA. Nucleic Acids Symp Ser (Oxf).2005; (49):5-6.

    First synthesis of 2’,4’-BNANC [NH] and [NMe]. 
    Hybridization.
    Nuclease stability
    Duplex-Formation RNA and DNA

    Tm Values of the 2’,4’-BNANC- Modified Oligonucleotides with Complementary ssRNA


     Tm=(ΔTm /modification) (°C)

    oligonucleotides T=        2’,4’-BNANC
      [NH] [NMe]
    d(GCGTTTTTTGCT) 45 45
    d(GCGTTTTTTGCT) 51 (+6.0) 50 (+5.0)
    d(GCGTTTTTTGCT) 61 (+5.3) 59 (+4.7)
    d(GCGTTTTTTGCT) 83 (+ 6.3) 80 (+5.8)
    Conditions: 100 mM NaCl, 10 mM sodium phosphate pH 7.2. 4 µM complementary strands.
    Target dsDNA: 5’-d(AGCAAAAAACGC)-3’
    Conclusion:
    Excellent nuclease resistance and strong hybridization ability against complementary strand
    Duplex formation

    2006:Rahman SM, Seki S, Utsuki K, Obika S, Miyashita K, Imanishi T.; High-affinity RNA mimicking binding of 2',4'-BNANC towards complementary strands: a comparative study with 2',4'-BNA/LNA. 2006 Nucleic Acids Symposium Series No. 50 195–196.
    2',4'-BNANC duplex-forming ability towards a single-stranded RNA was similar to or slightly higher
    than that of 2',4'-BNA(LNA) and the overall triplex-forming ability against a double-stranded DNA was also better than that of 2',4'-BNA(LNA). 2',4'-BNANC exhibited higher RNA selectivity than 2',4'-BNA.

    Tm values of both 2',4'-BNANC (N-H) and 2',4'-BNANC (N-Me) modified oligonucleotides were
    increased by 6 °C and 5°C, respectively, by a single modification.

    These results of duplex-forming affinity is similar to that of 2',4' -BNA/LNA modified oligonucleotide.
    Increasing the number of modifications from one to three, Trn values further increased and the T;n per modification (~Tm) was slightly higher than that of 2',4'-BNA/LNA.

    Binding affinity against a complementary RNA strand by 2',4'-BNANC modified oligonucleotides was similar to or slightly higher than that of 2' ,4'-BNA/LNAmodified oligonucleotides.

    Trn values by the modified oligonucleotides indicate that 2' ,4' -BNANC modified oligonucleotides
    possessed RNA selective binding affinity. 2',4' -BNANC (N-Me) modified oligonucleotides offered highestRNA selectivity amongst the modified oligonucleotides.

    Tm values of 2',4'-BNANC and 2',4'-BNA/LNA modified oligonucleotides against complementary ssRNA and ssDNA


    Tm (ΔTm /modification) (°C)
    Oligonucleotides RNA DNA
    d(GCGTTTTTTGCT) 45 50
    d(GCGTTXTTTGCT) 51 (+6.0) 51 (+1.0)
    d(GCGTTYTTTGCT) 50 (+5.0) 49 (-1.0)
    d(GCGTTZTTTGCT) 52 (+7.0) 53 (+3.0)
    d(GCGXTXTXTGCT) 64 (+6.3) 55 (+1.7)
    d(GCGYTYTYTGCT) 63 (+6.0) 50 (+0)
    d(GCGZTZTZTGCT) 62 (+5.7) 56 (+2.0)

    Note: X, Y, Z represent modification of the natural oligonucleotide by 2',4'-BNANC (N-H), 2',4'-BNANC (N-Me) and 2',4'-BNA/LNA thymine monomers, respectively

    Conditions:100 mM NaCI, 10 mM sodium phosphate buffer (pH 7.2), using 4 µM complementary strand; scan rate 0.5 °C/min (from 5 to 90 °C).

    Target sequence:
    5'
    r(AGCAAAAACGC)-3'   for RNA
    5'-d(AGCAAAAACGC)-3' for DNA

    CONCLUSION

    2',4'-BNANC monomers showed extraordinarily high binding affinity towards the complementary strands which is similar to or slightly higher than that of the corresponding oligonucleotides modified by 2',4'-BNA/LNA.

    2',4'-BNANC offers a site on the bridged structure (N atom) for further functionalization e.g. with fluorescence or DNA cleavage activators. 2007:  Kazuyuki Miyashita, S. M. Abdur Rahman, Sayori Seki, Satoshi Obikaab and Takeshi Imanishi;   N-Methyl substituted 2’,4’-BNANC: a highly nuclease-resistant nucleic acid analogue with high-affinity RNA selective hybridization.  Chem. Commun., 2007, 3765–3767.

    2', 4'-BNA(NC)[N-Me] in comparison to 2',4'-BNA (LNA), have similarly

    high RNA affinity,
    better RNA selectivity and
    muchhigher resistance to nuclease degradation.
    Good for antisense approaches.

    BNANCNME2 BNANCNME3

    Change in melting temperature (ΔTm) of modified oligonucleotides (7–9 and 11–13) relative to the reference oligonucleotide 15, 5’-d(GCGTTTTTTGCT)-3’. The Tm values of the duplexes formed by 15 with complementary DNA and RNA were 50 and 45 °C. Tm values were obtained from the maxima of the first derivatives of the melting curves (at 260 nm).

    Conditions: 4 mM strands solution in 10 mM sodium phosphate buffer (pH 7.2) containing 100 mM
    NaCl.

    Target sequences:                    DNA, 5’-d(AGCAAAAAACGC)-3’
                                                             RNA, 5’-r(AGCAAAAAACGC)-3’

    2007:Rahman SM, Seki S, Utsuki K, Obika S, Miyashita K, Imanishi T .; 2',4'-BNA(NC): a novel bridged nucleic acid analogue with excellent hybridizing and nuclease resistance profiles. Nucleosides Nucleotides Nucleic Acids.2007;26(10-12):1625-8.

    Abstract: Oligonucleotides modified with 2 ',4 '-BNA(NC) (N-H)/(N-Me) monomers exhibited excellent hybridizing and nuclease resistance properties. Duplex and triplex thermal stabilities were greatly enhanced by incorporating 2',4'-BNA(NC) (N-H) and (N-Me) monomers and nuclease resistance was tremendously higher than that of natural oligonucleotide.

    UV melting temperatures (Tm)Values of the 2’,4’-BNANC- Modified Oligonucleotides with Complementary ssRNA (Duplex Formation{DF}) and Triplex Formation (TFO)

      oligonucleotides
    5’ to 3’
    Tm(°C)
    DF
    ΔTm(°C)
    DF
    ΔTm(°C)/mod.
    DF
    Tm(°C)
    TFO
    ΔTm(°C)
    TFO
    DNA-1 d(GCGTTTTTTGCT) 45 - -    
    DNA-2 d(TTTTTmCTTTmCTmCTmCT)       33  
    ON-1 d(GCGTTXTTTGCT) 51 +6.0 +6.0    
    ON-2 d(GCGTTYTTTGCT) 50 +5.0 +5.0    
    ON-3 d(GCGXTXTXTGCT) 64 +19 +6.3    
    ON-4 d(GCGYTYTYTGCT) 63 +18 +6.0    
    ON-5 d(TTTTTmCTXTmCTmCTmCT)       44 +11.0
    ON-6 d(TTTTTmCTYTmCTmCTmCT)       38 +5.0

    (X = 2’,4’-BNANC (N-H) thymine and Y = 2’,4’-BNANC (N-Me) thymine monomers, respectively; mC=
    5-methylcytidine) . Conditions: 100 mM NaCl, 10 mM sodium phosphate pH 7.2. 4 µM complementary strands.

    Target strands used for duplex and triplex forming experiments were

    RNA:   5_-r(AGCAAAAACGC)-3
    DNA:   5_-d(GCTAAAAAGAAAGAGAGATCG)-3
    DNA:   3_-d(CGATTTTTCTTTCTCTCTAGC)-5

    CONCLUSION

    2’,4’-BNANC (N-H) and (NMe) greatly enhance duplex and triplex stability against ssRNA and dsDNA.

    The Nuclease resistance property of both 2’,4’-BNANC (N-H) and (N-Me) modified oligonucleotides was very much higher than that of natural oligonucleotide.

    2’4’-BNANC (N-H) analogue offers a site (N-atom) on the bridged structure for further functionalization with various functional moieties (such as florescence, DNA cleavage activator) which would provide versatile applications in genomics.

    2007:  S. M. Abdur Rahman, Sayori Seki, Satoshi Obika, Sunao Haitani, Kazuyuki Miyashita, and Takeshi Imanishi; Highly Stable Pyrimidine-Motif Triplex Formation at Physiological pH Values by a Bridged Nucleic Acid Analogue. Angew. Chem. Int. Ed. 2007, 46, 4306 –4309.

    Tm values of triplexes containing 2,4,-BNANC[NH] (bold red), 2’,4’-BNA(LNA) (bold blue), and ENA (bold black).[a,b]

     
     
    TFO Sequence (5’…3’) Tm [°C] ΔTm [°C] ΔTm/mod
    ON-0 TTTTTmCTTTmCTmCTmCT 33
    ON-1 TTTTTmCTTTmCTmCTmCT 44 +11 +11.0
    BNA-1 TTTTTmCTTTmCTmCTmCT 44 +11 +11.0
    ENA-1 TTTTTmCTTTmCTmCTmCT 42 +9 +9.0
    ON-2 TTTTTmCTTTmCTmCTmCT 60 +27 +9.0
    BNA-2 TTTTTmCTTTmCTmCTmCT 59 +26 +8.7
    ENA-2 TTTTTmCTTTmCTmCTmCT 56 +23 +7.7
    ON-3 TTTTTmCTTTmCTmCTmCT 59 +26 +8.7
    BNA-3 TTTTTmCTTTmCTmCTmCT 52 +19 +6.3
    ENA-3 TTTTTmCTTTmCTmCTmCT 57 +24 +8.0
    ON-4 TTTTTmCTTTmCTmCTmCT 58 +25 +6.3
    BNA-4 TTTTTmCTTTmCTmCTmCT 57 +24 +6.0
    ENA-4 TTTTTmCTTTmCTmCTmCT 57 +24 +6.0
    ON-5 TTTTTmCTTTmCTmCTmCT 64 +31 +6.2
    BNA-5 TTTTTmCTTTmCTmCTmCT 65 +32 +6.4
    ENA-5 TTTTTmCTTTmCTmCTmCT 58 +25 +5.0
    ON-6 TTTTTmCTTTmCTmCTmCT 78 +45 +6.4
    BNA-6 TTTTTmCTTTmCTmCTmCT 67 +34 +4.9
    ENA-6 TTTTTmCTTTmCTmCTmCT 72 +39 +5.6
    ON-7 TTTTTmCTTTmCTmCTmCT 80 +47 +3.1
    BNA-7 TTTTTmCTTTmCTmCTmCT <5 <-28 <-2
    [a] Target duplex:     5’-d(GCTAAAAAGAAAGAGAGATCG)-3’/  
    3’-d(CGATTTTTCTTTCTCTCTAGC)-5’ ;  

    underlined portion indicates the target site for triplex formation.

    [b] Conditions: 7 mm Na2HPO4 buffer solution containing 140 mm KCl; strand concentration=1.5 mm; scan rate 0.5 °C/min. Tm = melting temperatures, ΔTm = changes in melting temperature, ΔTm/mod=changes in melting temperature per single modification; mC = 5-methylcytidine.

    Conclusion:   TFOs composed of 2’,4’-BNANC forms highly stable pyrimidine-motif triplexes at physiological pH values.

    Triplex-forming ability is higher than that of 2’,4’-BNA/LNA- and ENA-modified TFOs.

    These TFOs eliminate the requirement of placing alternating DNA monomers for optimum efficacy.

    2007:  Satoshi Obika, Masaharu Tomizu, Yoshinori Negoro, Ayako Orita, Osamu Nakagawa, and Takeshi Imanishi;  Double-Stranded DNA-Templated Oligonucleotide Digestion Triggered by Triplex Formation. ChemBioChem 2007, 8, 1924 – 1928. Note: Triplex triggered cleavage of oligonucleotides.

    Bond cleavage reactions promoted by hybridization with DNA templates comprise a new class of DNA-templated organic synthesis (DTS) and should be useful for novel DNA sensing technologies.

    Oligonucleotides with P3’->N5’ phosphoramidate linkages can be cleaved at the phosphoramidate bond under mild acidic conditions.

    dsDNA sensing probes

    BNANCNME4

    Effect of triplex formation on cleavage of oligonucleotides 1–3.[a]

    5’-d(TTTTTCTXTCTCTCT)-3’
    oligonucleotide 1  [X= 5’-amino-DNA-T]
    oligonucleotide 2  [X = 5’-amino-2’4’-BNA-T(R = H)]
    oligonucleotide 3  [X = 5’-amino-3’,5’-BNA-T]
    oligonucleotide 4  [X = 5’-amino-2’4’-BNA-T(R = Me)]

    5’-d(GCTAAAAAGAYAGAGAGATCG)-3’
    5’-d(CGATTTTTCTZTCTCTCTAGC)-3’

    dsDNA target (perfect match)            5 [YZ = AT]
    dsDNA target (mis match)                 6 [YZ = TA], 7 [YZ = GC], 8 [YZ = CG]

    C = 2’-deoxy-5-methylcytidine

      oligonucleotide 1 oligonucleotide 2 oligonucleotide 3
      60 min 240 min 60 min 240 min 60 min 240 min
    + target 5 50% 4% 42% 2% 25% 0%
    - target 5 83% 48% 95% 74% 50% 3%

     [a] Oligonucleotides 1–3 were treated at pH 3 for the indicated time period in the presence or absence of a
    perfectly matched dsDNA target, 5. The percentage of remaining intact oligonucleotide was determined by
    HPLC.

    BNANCNME6

    dsDNA sensing by the acid-mediated phosphoramidate cleavage (APAC) probes.
     
    A) Sequences of the APAC probes and the dsDNA targets. F, TAMRA-dT; Q, BHQ-2-dT; C, 2’-deoxy-5-methylcytidine; and X, 5’-amino-2’,4’-BNA-T (R=Me).
    B) Emission spectra of the APAC probe A in the absence (left) or presence (right) of the fully matched dsDNA target A. APAC probe A was incubated in an acidic buffer (pH 4) at 408C. After 0 (red), 10 (green), 30 (blue), and 60 min (yellow), the reaction mixture was neutralized and the emission spectrum was measured with excitation at 545 nm.
    C) Images of the fluorescence of APAC probes A–C in the presence or absence of the dsDNA targets. The samples were incubated for 10 min at pH 4.0 and 408C, neutralized, and photographed.

    2008: Masayasu Kuwahara, Satoshi Obika, Jun-ichi Nagashima, Yuki Ohta, Yoshiyuki Suto, Hiroaki Ozaki, Hiroaki Sawai and Takeshi Imanishi; Systematic analysis of enzymatic DNA polymerization using oligo-DNA templates and triphosphate analogs involving 2’,4’-bridged nucleosides. Nucleic Acids Research, 2008, Vol. 36, No. 13 4257–4265.

    SELEX, DNA Aptamer, DNAzymes

    Five types of thermostable DNA polymerases used Taq, Phusion HF, Vent(exo-), KOD Dash and
    KOD(exo-), the KOD Dash and KOD(exo-) DNA polymerases could smoothly read through the modifiedtemplates containing 2’-O,4’-C-methylene-linked nucleotides at intervals of a few nucleotides, even at standard enzyme concentrations for 5 min.

    KOD(exo-) DNA polymerase was found to be far superior to the Vent(exo-) DNA polymerase in accurate incorporation of nucleotides.

    Successive incorporation of 2’,4’-bridged nucleotides into extending strands using 2’,4’-bridged nucleoside-5’-triphospates was much more difficult.

    These data indicate that the sugar modification would have a greater effect on the polymerase reaction when it is adjacent to the elongation terminus than when it is on the template as well, as in base modification.

    CONCLUSION

    KOD Dash DNA polymerase is suitable for enzymatic production of modified DNA containing base-modified nucleotides.

    Using this DNA polymerase, we prepared a modified DNA library involving C5-modified thymidine and successfully screened modified DNA aptamers bound to sialyllactose, R-isomer of thalidomide derivative, and so on by SELEX.

    Thus, KOD Dash DNA polymerase could accept a broad range of nucleotide modifications and might be best suited for enzymatic preparation of functional modified DNA.

    The BNA templates containing sequences of seven successive 2’,4’-bridged nucleotides Ks, Ls and Ms could not be completely transcribed by any DNA polymerases used; yields of longer elongated products decreased in the order of steric bulkiness of the modified sugars.

    Successive incorporation of bridged nucleotides into extending strands using triphosphates KTP (LNA), LTP(O-.-O) and MTP(BNANC[NH]) were much more difficult.

    KOD Dash and KOD(exo-) DNA polymerases could smoothly read through the BNA templates containing Ks or KAs at intervals of three nucleotides, two nucleotides and one nucleotide, respectively, and produce the corresponding complimentary natural DNA strand even under standard enzyme concentrations.

    Vent(exo-) DNA polymerase also read through these BNA templates; however, kinetic study indicates that KOD(exo-) was found to be far superior to Vent(exo-) in accurate incorporation of nucleotides.

    2008:  Satoshi Obika, S. M. Abdur Rahman, Bingbing Song, Mayumi Onoda, Makoto Koizumi, Koji Morita, Takeshi Imanishi; Synthesis and properties of 3’-amino-2’,4’-BNA, a bridged nucleic acid with a N3’->P5’ phosphoramidate linkage. Bioorganic & Medicinal Chemistry 16 (2008) 9230–9237.

    Oligonucleotides containing the 3’-amino-2’,4’-BNA residue form highly stable duplexes and triplexes with single-stranded DNA (ssDNA), single-stranded RNA (ssRNA), and double-stranded DNA (dsDNA) targets, with the average increase in melting temperature (Tm) against ssDNA, ssRNA and dsDNA being +2.7 to +4.0 °C, +5.0 to +7.0 °C, and +5.0 to +11.0 °C.

    Comparable to LNAs.

    Oligonucleotide modified with a single 3’-amino-2’,4’-BNA thymine residue showed extraordinarily high resistance to nuclease degradation, much higher than that of LNAs and substantially higher even than that of 3’-amino-DNA and phosphorothioate oligonucleotides.

    BNANCNME7

    Duplex Formation

    Tm values of duplexes formed by 3’-amino-2’,4’-BNA oligonucleotides with ssDNA and ssRNA a,b


    Tm (ΔTm /modification) (°C) 
       _________________________
    Oligonucleotides,  5’ to 3’ ssDNA     ssRNA
    T = 3’-amino-2’,4’-BNA-T  
    GCGTTTTTTGCT 47 45
    GCGTTTTTTGCT 51 (+4.0) 52 (+7.0)
    GCGTTTTTTGCT 53 (+2.0) 63 (+6.0)
    GCGTTTTTTGCT 53 (+2.0) 61 (+5.3)
    GCGTTTTTTGCT 63 (+2.7) 75 (+5.0)
    t = 2’,4’-BNA-T (LNA)   
    GCGTTtTTTGCT 53 (+6.0) 52 (+7.0)c
    GCGtTtTtTGCT 56 (+3.0) 62 (+5.7)c
    GCGTTtttTGCT 54 (+2.3) 60 (+5.0)c
    GCGttttttGCT 67 (+3.3) 80 (+5.8)d

    a Targets:
    ssDNA,           5’-d(AGCAAAAAACGC)-3’;
    ssRNA,           5’-r(AGCAAAAAACGC)-3’ .

    b Conditions: 10 mM sodium phosphate buffer (pH 7.2) containing 100 mM NaCl;
    strand concentration = 4 µM. T = 3’-amino-2’,4’-BNA-T, t = 2’,4’-BNA-T (LNA).
    c Data from Obika et al., 2003. d Data from Imanishi and Obika, 1999.

    Duplex thermal stability further improved upon increasing the number of modifications.
    Incorporating three 3’-amino-2’,4’-BNA residues either consecutively or separated by natural DNA units resulted in duplexes with very high thermal stability.

    The most prominent enhancement in thermal stability was observed in duplexes formed with complementary ssRNA.

    A modified oligonucleotide containing six consecutive 3’-amino-2’,4’-BNA residues also formed duplexes with ssRNA and ssDNA with remarkably improved thermal stability.

    Triplex Formation

    Tm values of triplexes formed by 3’-amino-2’,4’-BNA oligonucleotides with dsDNA a,b

    _________________________________________________________________________________
    Tm (ΔTm /modification) (°C)
    _______________________
    Oligonucleotides,  5’ to 3’  Targets - MgCl2 + MgCl2

     

    TTTTTmCTTTmCTmCTmCT d(GCTAAAAAGAAAGAGAGATCG)-3’
    d(CGATTTTTCTTTCTCTCTAGC)-5’
    32 44
    TTTTTmCTTTmCTmCTmCT d(CGATCTCTCTTTCTTTTTAGCCCCCGCTAAA
    AAGAAAGAGAGATCG)-3’ hairpin
    32 39
    TTTTTmCTTTmCTmCTmCT d(GCTAAAAAGAAAGAGAGATCG)-3’
    d(CGATTTTTCTTTCTCTCTAGC)-5’
    44 (+11) 55 (11)
    TTTTTmCTTTmCTmCTmCT d(CGATCTCTCTTTCTTTTTAGCCCCCGCTAAA
    AAGAAAGAGAGATCG)-3’ hairpin
    59 (+5.4) 71 (+6.4)
    TTTTTmCTtTmCTmCTmCT d(GCTAAAAAGAAAGAGAGATCG)-3’
    d(CGATTTTTCTTTCTCTCTAGC)-5’
    44 (+11) 57 (+13)
    TTTtTmcTtTmcTmcTmCT d(CGATCTCTCTTTCTTTTTAGCCCCCGCTAAA
    AAGAAAGAGAGATCG)-3’ hairpin
    60 (+5.6) 72 (+6.6)

    Conditions: 7 mM sodium phosphate buffer (pH 7.0) containing 140 mM KCl in the absence or the presence of MgCl2 (10 mM); strand concentration = 1.5 µM. T = 3’-amino-2’,4’-BNA-T, mC = 3’-amino-2’,4’-BNA-mC, t = 2’,4’-BNA-T, mc = 2’,4’-BNA-mC.

    Sequence specific triplex by 3’-amino-2’,4’-BNA modified TFO a

    _________________________________________________________________________________
    TmTm (ΔTm – Tm(mismatch) – Tm (match)) (°C)
    _____________________________________________________________
    Oligonucleotides, 5’ to 3’ X:Y =A:T (match) G:C C:G T:A
    TTTTTmCTTTmCTmCTmCT d(GCTAAAAAGAXAAAGAGATCG)
    d(CGATTTTTCYTTCTCTCTAGC)
    44 20(-24) 25(-19) 17(-25)
    TTTTTmCTTTmCTmCTmCT d(GCTAAAAAGAXAAAGAGATCG)
    d(CGATTTTTCYTTCTCTCTAGC)
    55 31(-24) 32 (-23) 16 (-39)
    TTTTTmCTtTmCTmCTmCT d(GCTAAAAAGAXAAAGAGATCG)
    d(CGATTTTTCYTTCTCTCTAGC)
    57 31 (-26) 35 (-22) 16 (-41)

    T = 3’-Amino-2’,4’-BNA; t = LNA

    a Conditions: 7 mM sodium phosphate buffer (pH 7.0) containing 140 mM KCl and 10 mM MgCl2; strand concentration = 1.5 µM.

    Conclusion:

    3’-amino-2’,4’-BNA oligonucleotides show better RNA selective binding affinity than LNA oligos.

    Mismatch discrimination of 3’-amino-2’,4’-BNA is similar to that of LNA.

    Nuclease resistance of 3’-amino-2’,4’-BNA is excellent. Much higher than that of natural and LNA oligonucleotides, and substantially higher even than that of 3’-amino-DNA and phosphorothioate oligonucleotides.

    Useful for antisense and antigene applications.

    2008:S. M. Abdur Rahman, Sayori Seki, Satoshi Obika, Haruhisa Yoshikawa, Kazuyuki Miyashita, and Takeshi Imanishi;   Design, Synthesis, and Properties of 2’,4’-BNANC: A Bridged Nucleic Acid Analogue.  J. AM. CHEM. SOC. 2008, 130, 4886-4896.

    Very high target affinity
    BNANC[NMe] has high RNA affinity and better RNA selectivity than LNA
    BNANC[NH] has enhanced triplex formation ability
    Fully modified BNANC[NH] oligonucleotide forms stable triplex at neutral pH.
    BNANC[NH] oligonucleotide has dramatically improved resistance to nuclease degradation.
    Antisense applications
    BNANC[NH] oligonucleotide very good for antigene applications

    2008:  Sasaki K, Rahman SM, Obika S, Imanishi T, Torigoe H.; Promotion of triplex formation by 2'-O,4'-C-aminomethylene bridged nucleic acid (2',4'-BNA NC) modification. Nucleic acids symposium series (2004) : 52 2008 pg 419-20.

    Abstract:  We examined the effect of 2'-O,4'-C-aminomethylene bridged nucleic acid (2',4'-BNA(NC)) backbone modification of triplex-forming oligonucleotide (TFO) on the pyrimidine motif triplex formation at neutral pH, a condition where pyrimidine motif triplexes are unstable. The melting temperature of the pyrimidine motif triplex at pH 6.8 with 2',4'-BNA(NC) modified TFO was significantly higher than that observed with unmodified TFO. The 2',4'-BNA(NC) modification of TFO increased the thermal stability of the pyrimidine motif triplex at neutral pH. The present results certainly support the idea that the 2',4'-BNA(NC) backbone modification of TFO could be a key chemical modification and may eventually lead to progress in therapeutic applications of the antigene strategy in vivo.

    BNANC[NH] forms TFOs.

    2010:  Yuuya Kasahara, Shunsuke Kitadume, Kunihiko Morihiro, Masayasu Kuwahara, Hiroaki Ozaki, Hiroaki Sawai, Takeshi Imanishi, Satoshi Obika;  Effect of 3’-end capping of aptamer with various 2’,4’-bridged nucleotides: Enzymatic post-modification toward a practical use of polyclonal aptamers. Bioorganic & Medicinal Chemistry Letters 20 (2010) 1626–1629.

    The capping of the 3′-ends of thrombin binding aptamers (TBAs) with bridged nucleotides increased the nuclease resistances and the stabilities in human serum. The binding abilities of the aptamers were not affected by the capping. The capping could be simply executed via a one step enzymatic process using 2′,4′-bridged nucleoside 5′-triphosphate and terminal deoxynucleotidyl transferase.

    BNANCNME8 BNANCNME9 BNANCNME10

    2010: S. M. Abdur Rahman, Hiroyuki Sato, Naoto Tsuda, Sunao Haitani, Keisuke Narukawa, Takeshi Imanishi, Satoshi Obika; RNA interference with 2’,4’-bridged nucleic acid analogues. Bioorganic & Medicinal Chemistry 18 (2010) 3474–3480.

    siRNA to inhibit firefly luciferase expression in CHO-luc cells.

    2011:  Torigoe H,Rahman SM, Takuma H, Sato N, Imanishi T, Obika S, 2'-O,4'-C-aminomethylene-bridged nucleic acid modification with enhancement of nuclease resistance promotes pyrimidine motif triplex nucleic acid formation at physiological pH.Chemistry (Weinheim an der Bergstrasse, Germany) 17:9 2011 Feb 25 pg 2742-51.

    Abstract: Due to the instability of pyrimidine motif triplex DNA at physiological pH, triplex stabilization at physiological pH is crucial in improving its potential in various triplex-formation-based strategies in vivo, such as gene expression regulation, genomic DNA mapping, and gene-targeted mutagenesis. To this end, we investigated the thermodynamic and kinetic effects of our previously reported chemical modification, 2'-O,4'-C-aminomethylene-bridged nucleic acid (2',4'-BNA(NC)) modification of triplex-forming oligonucleotide (TFO), on triplex formation at physiological pH. The thermodynamic analyses indicated that the 2',4'-BNA(NC) modification of TFO increased the binding constant of the triplex formation at physiological pH by more than 10-fold. The number and position of the 2',4'-BNA(NC) modification in TFO did not significantly affect the magnitude of the increase in the binding constant. The consideration of the observed thermodynamic parameters suggested that the increased rigidity and the increased degree of hydration of the 2',4'-BNA(NC)-modified TFO in the free state relative to the unmodified TFO may enable the significant increase in the binding constant. Kinetic data demonstrated that the observed increase in the binding constant by the 2',4'-BNA(NC) modification resulted mainly from the considerable decrease in the dissociation rate constant. The TFO stability in human serum showed that the 2',4'-BNA(NC) modification significantly increased the nuclease resistance of TFO. Our results support the idea that the 2',4'-BNA(NC) modification of TFO could be a key chemical modification to achieve higher binding affinity and higher nuclease resistance in the triplex formation under physiological conditions, and may lead to progress in various triplex-formation-based strategies in vivo.

    BNANC[NH] froms TFOs

    2011: Tsuyoshi Yamamoto, Moeka Nakatani, Keisuke Narukawa & Satoshi Obika; Antisense drug discovery and development Future Med. Chem. (2011) 3(3), 339–365. Overview including BNANCs.

    Antisense drug discovery
    Antisense drug development
    Antisense medicinal chemistry
    Antisense in clinical trial

    Translational arrest:     oligonucleotide (ON); mRNA degradation:   RNase H recruitment; Splice switching: Splice switching oligonucleotide (SSO)

    2012:  Tsuyoshi Yamamoto, Mariko Harada-Shiba, Moeka Nakatani, Shunsuke Wada, Hidenori Yasuhara, Keisuke Narukawa, Kiyomi Sasaki, Masa-Aki Shibata, Hidetaka Torigoe, Tetsuji Yamaoka, Takeshi Imanishi and Satoshi Obika;  Cholesterol-lowering Action of BNA-based Antisense Oligonucleotides Targeting PCSK9 in Atherogenic Diet-induced Hypercholesterolemic Mice Molecular Therapy–Nucleic Acids (2012) 1, e22; oi:10.1038/mtna.2012.16.

    BNA-based antisense therapeutics can be used to successfully inhibit hepatic PCSK9 expression which resulted in a strong reduction of the serum LDL-C levels of mice.
    PCSK9 is a potential therapeutic target for hypercholesterolemia.
    BNA-based antisense oligo-nucleotides (AONs) induced a cholesterol-lowering action in hypercholesterolemic mice. 

     

    0 0

    Buffers used in BNA experiments

    Application Concentration Buffer Reference
    Antisense BNAs 1 micomolar (1 µM) 20 mM sodium phosphate, 100 mM NaCl pH 7.2 Obika et al., 2001
    Duplex formation Duplex concentration 4 micromolar (µM) 100 mM NaCl, 10 mM sodium phosphate pH 7.2 Obika et al., 1998
    Duplex formation 4 microM (µM) per strand 100 mM NaCl, 10 mM sodium phosphate pH 7.2 Imanishi and Obika 2002
    Duplex formation 4 micromolar (µM) 100 mM NaCl, 10 mM sodium phosphate pH 7.2 Miyashita et al., 2007
    Triplex formation > 0.2 µM

    140 mM KCl, 10 mM MgCl2, 7 mM sodium phosphate pH 7.0

    50 mM Tris-acetate, 100 mM MgCl2 pH 7.0

    10 mM sodium cacodylate/cacodylic acid pH 6.8 200 mM NaCl, 20 mM MgCl2

    Hari et al., 2003


    Torigoe et al., 2011

    RNAi

    1 µM

    Transfection:

    0.2 to 20 nM

    100 mM NaCl, 10 mM sodium phosphate pH 7.2

    Transfection: Lipofectamine 2000 in OPTI-MEM buffer

    Rahman et al. 2010,

    Yamamoto et al. 2012

    Example of experimental conditions for FISH

    Table 1: Different conditions can be tried with FISH technique using BNA/DNA mixmers as probes
    Probe amount

    150 ng (20 pmoles)

    100 ng (13.4 pmoles)

    75 ng (10 pmoles)

    50 ng (6.4 pmoles)

    Denaturation

    Separate denaturation of slide and probe

    at 75 XoC for 5 min

    Simultaneous denaturation at 75 oC

    No denaturation

    Hybridization mixture

    50% formamide/2xSSC/10% dextran (pH 7.0)

    2xSSC/10% dextran sulfate (pH 7.0)

    Hybridization time

    Overnight

    5 h

    3 h

    1 h

    30 min

    Hybridization temperature

    RT

    37 oC

    55 oC

    60 oC

    72 oC

    Post wash

    Normal FISH wash

    60 oC 50% formamide

    No formamide wash

    The saline-sodium citrate (SSC) buffer is used as a hybridization buffer, to control stringency for washing steps in protocols for Southern blotting, in situ hybridization, DNA Microarray or Northern blotting. 20X SSC may be used to prevent drying of agarose gels during a vacuum transfer.

    A 20X stock solution consists of 3 M sodium chloride and 300 mM trisodium citrate (adjusted to pH 7.0 with HCl).

    A 2X SSC buffer consists of 0.3 M sodium chloride and 30.0 mM trisodium citrate (adjusted to pH 7.0 with HCl).

    Procedure

    FISH is carried out as described in Table 1. The amount of probe can be varied between 6.4, 10, 13.4 and 20 pmoles.

    Denaturation of the target DNA and the probe is performed at 75 oC for 5 min either separately using 70% formamide or simultaneously under the coverslip in the presence of hybridization mix containing 50% formamide. In addition, effect of no denaturation is also tested. Two alternative hybridization mixtures can be used: 50% formamide/2xSSC (pH 7.0) / 10% dextran sulphate or 2xSSC (pH 7.0)/10% dextran sulphate.

    Hybridization times include 30 min, 1, 2, 3 h and overnight. Hybridization temperatures include: 37, 55, 60 and 72 oC. Post washing is either done as for standard FISH, or with 50% formamide/2xSSC at 60 oC, or without formamide. Hybridization signals with biotin labeled BNA/DNA mixmers are visualized indirectly using two layers of fluorescein labeled avidin linked by a biotinylated anti-avidin molecule, which amplifies the signal 8–64 times. The hybridization of Cy3 labeled molecules, however, is visualized directly after a short washing procedure.

    Slides can be mounted in Vectashield containing 40-60-diamidino-2-phenylindole (DAPI).

    The whole procedure is carried out in the dark. The signals can be visualized using a Leica DMRB epifluorescence microscope equipped with a SenSys charge-coupled device camera (Photometrics, Tucson, AZ), and IPLAB Spectrum Quips FISH software (Applied Imaging international Ltd, Newcastle, UK) within 2 days after hybridization.

    In general, twenty metaphases are analyzed after each hybridization experiment.

    Reference

    Silahtaroglu AN, Hacihanefioglu S, Guven GS, Cenani A, Wirth J, Tommerup N, Tumer Z. Not para-, not peri-, but centric inversion of chromosome 12. J Med Genet 1998;35(8):682–4.


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    What are lncRNAs and lincRNAs

    Recent discoveries increasingly indicate that only a small percentage, approximately 7%, of disease-associated single nucleotide polymorphisms (SNPs) are located in protein-coding regions whereas the remaining 93% are located in gene regulatory regions or in intergenic regions. Therefore our understanding of how genetic variations control the expression of non-coding RNAs tissue-specific will have far reaching implications for how diseases will need to be treated in the future.

    Advances made in high-throughput RNA sequencing technologies have provided scientists an expanded view of the complexity of a genome and it has now become clear that most of the human genome is transcribed to produce not only protein-coding transcripts but also large numbers of non-coding RNAs (ncRNAs) of different size. By now many types of RNA have been well characterized. These include short ncRNAs, microRNAs, small interfering RNAs, and piwi-interacting RNAs.

    However, it was discovered that the large intergenic non-coding RNAs (lincRNAs) make up most of the long ncRNAs. LincRNAs are non-coding transcripts of more than 200 nucleotides long; they have an exon-intron-exon structure, similar to protein-coding genes, but do not encompass open-reading frames and do not code for proteins. More than 8,000 lincRNAs have been descript recently and it is thought that lincRNAs are the largest subclass of RNAs originating from the non-coding transcriptome in humans.

    According to Cabili et al. (2011) “Large intergenic non-coding RNAs (lincRNAs) are the largest class of non-coding RNA molecules in the human genome. Many genome-wide association studies (GWAS) have mapped disease-associated genetic variants (SNPs) to, or in, the vicinity of such lincRNA regions. At this point in time it is not clear how these SNPs can affect the disease.” Cabili et al. tested whether SNPs were also associated with the lincRNA expression levels in five different human primary tissues and observed that there is a strong genotype-lincRNA expression correlation that is tissue-dependent. Many of the observed lincRNA cis-eQTLs are disease- or trait-associated SNPs. Their results suggested that lincRNA-eQTLs represent a novel link between non-coding SNPs and the expression of protein-coding genes, which can be exploited to understand the process of gene-regulation through lincRNAs in more detail.” Note that expression quantitative trait loci (eQTLs) are genomic loci that regulate expression levels of mRNAs or protein.

    Long non-coding RNAs are non-protein coding sequence transcripts that contain more than 200 nucleotides. Their size distinguishes lncRNAs from small regulatory RNAs such as microRNAs (miRNAs), short interfering RNAs (siRNAs), Piwi-interacting RNAs (piRNAs), small nucleolar RNAs (snoRNAs), short hairpin RNA (shRNA), and other short RNAs. Since the central dogma of gene expression is that DNA is transcribed into messenger RNAs that serve as the templates for protein synthesis lncRNAs where considered in the past as “junk” DNA or the “dark matter” of DNA. The discovery of extensive transcription of large RNA molecules that do not code for proteins provided a new perspective on the roles of RNA in gene regulation.

    New findings when studying multiple model systems suggest that lncRNAs form extensive networks of ribonucleoprotein (RNP) complexes with numerous chromatin regulators to target these enzymatic activities to the appropriate locations in the genome. Apparently lncRNAs can function as modular scaffolds to specify higher-order organization in RNP complexes and in chromatin states. Recent research in human transcriptome analysis showed that protein-coding sequences only account for a small portion of the genome transcripts. The majority of the human genome transcripts are non-coding RNAs now called long-non-coding RNAs (lncRNAs). Chen et al. in 2013 describe a long-non-coding RNA (lncRNA) and disease association database (LncRNADisease). This database is publicly accessible at http://cmbi.bjmu.edu.cn/lncrnadisease.

    Functions of lncRNAs

    Functions of lncRNAs

    In recent years a variety of regulatory paradigms for how long ncRNAs may function have been identified. LncRNAs may function by binding to DNA or RNA in a sequence specific manner or by binding to proteins. In contrast to miRNAs, lncRNAs appear not to operate by a common mode of action but can apparently regulate gene expression and protein synthesis in a number of ways.

    1. Transcription from an upstream noncoding promoter (yellow) can negatively or positively affect expression of the downstream gene (blue) by inhibiting RNA polymerase II recruitment or inducing chromatin remodeling, respectively.

    2. An antisense transcript (purple) is able to hybridize to the overlapping sense transcript (blue) and block recognition of the splice sites by the spliceosome. This results in an alternatively spliced transcript.

    3. Hybridization of the sense and antisense transcripts can allow Dicer to generate endogenous siRNAs.

    4. Binding of the noncoding transcript (green) to specific protein partners can modulate the activity of the protein or serve as a structural component that allows a larger RNA–protein complex to form, or alter where the protein localizes in the cell.

    5. Long ncRNAs (red) can be processed to yield small RNAs, such as miRNAs, piRNAs, and other less well-characterized classes of small transcripts.
    Classification of lncRNA

    lncRNAs can be classified into the following locus biotypes based on their location with respect to protein-coding genes:

    1. Intergenic lncRNA: Intergenic lncRNAs are transcribed intergenetically from both strands.
    2. Intronic lncRNA:Intronic lncRNAs are entirely transcribed from introns of protein-coding genes.
    3. Sense lncRNA: Sense lncRNAs are transcribed from the sense strand of protein-coding genes and contain exons from protein-coding genes that overlap with part of protein-coding genes or cover the entire sequence of a protein-coding gene through an intron.
    4. Antisense lncRNA :Antisense lncRNAs are transcribed from the antisense strand of the protein-coding genes that overlap with exonic or intronic regions or cover the entire protein-coding sequence through an intron.

    Further, it needs to be pointed out that presently there is still an uncertainty between comparative results. However, it has become clear that lncRNAs are emerging as regulatory elements of embryonic pluripotency, differentiation, and patterning of the body axis as well as promoting developmental transitions.

    Potential Functions and Molecular Mechanisms of lncRNAs

    Wang and Chang in 2011 illustrated the construction of complex functions by using combinations of archetypical molecular mechanisms of lncRNAs.

    Potential Functions and Molecular Mechanisms of lncRNAs

    Schematic diagram of the four archetypes of lncRNA mechanisms

    (Adapted from Wang, and Chang, Mol Cell. 2011)

    Archetype I : As Signals, lncRNA expression can faithfully reflect the combinatorial actions of transcription factors (colored ovals) or signaling pathways to indicate gene regulation in space and time.

    Archetype II : As Decoys, lncRNAs can titrate away transcription factors and other proteins away from chromatin, or titrate the protein factors into nuclear subdomains. A further example of decoys is lncRNA decoy for miRNA target sites (not shown on schematic).

    Archetype III : As Guides, lncRNAs can recruit chromatin modifying enzymes to target genes, either in cis (near the site of lncRNA production) or in trans to distant target genes.

    Archetype IV : As scaffolds, lncRNAs can bring together multiple proteins to form ribonucleoprotein complexes. The lncRNA-RNP may act on chromatin as illustrated to affect histone modifications. In other instances, the lncRNA scaffold is structural and stabilizes nuclear structures or signaling complexes.

    lncRNAs and disease and research

    lncRNAs are now implicated in a variety of diseases. Recent studies have shown that lncRNAs are differently expressed in various types of cancer including leukemia, breast cancer, hepatocellular carcinoma, colon cancer, and prostate cancer. In other diseases, such as cardiovascular diseases, neurological disorders and immune-mediated diseases, lncRNA appear to be dysregulated.

    Since lncRNA can be present in very low amounts, can overlap with coding transcripts on both strands and are often only found in the nucleus working with this type of RNAs can be very challenging. The table below list differences and similarities of mRNAs, lncRNAs and lincRNAs.

    A comparison of mRNAs, lncRNAs and lincRNAs
    mRNA lncRNA lincRNA
    Tissue-specific expression Tissue-specific expression Tissue-specific expression
    Form secondary structure Form secondary structure Form secondary structure
    Undergo post-translational processing such as 5’capping, polyadenylation, splicing Undergo post-translational processing such as 5’capping, polyadenylation, splicing Undergo post-translational processing such as 5’capping, polyadenylation, splicing
    Important in diseases and development Important in diseases and development Important in diseases and development
    Protein coding transcript Non-protein coding, regulatory function Non-protein coding, regulatory function
    Conserved between species Poorly conserved between species Poorly conserved between species
    Present in both nucleus and cytoplasm Predominantly in nucleus --
    Total 20-24,000 mRNAs Predict 3-100 fold more than mRNAs --
    Expression level: low to high Expression level: very low to moderate --
    Advantages of BNA-enhanced research for ncRNA

    Bridged nucleic acids (BNA3) are artificial bicyclic oligonucleotides that contain a six-membered bridged structure with a “fixed” C3’-endo sugar puckering. The bridge is synthetically incorporated at the 2’, 4’-position of the ribose to afford a 2’, 4’-BNA monomer. BNAs are structurally rigid oligo-nucleotides with increased binding affinities and stability.

    BNA monomers can be used for both primers and probes in real time quantitative polymerase chain reaction (RT-Q-PCR) assays. Compared to locked nucleic acids (LNAs) the substitution of DNA monomers with BNA monomers in oligonucleotides adds exceptional biological stability, resistance to nucleases and a significantly increased affinity to their complementary DNA targets.

    In addition, short, high affinity, BNA-enhanced qPCR primers can enhance the detection of low abundant targets. Furthermore, the specific placement of BNA monomer within the oligonucleotide probe allows to adjust the melting temperature (Tm) of the probe which may be important for qPCR analysis of overlapping transcripts.

    Applications of BNA oligonucleotides for lncRNA and lincRNA research
    • RNA isolation
    • Custom BNAs for qPCR assay development for mRNA and ncRNA
    • Custom BNA detection probes for ncRNAs
    • BNA long RNA Gapmer Antisense Oligonucleotides
    • Custom BNA Oligonucleotides
    Biosynthesis Incorporated

    has 28 years experience in the analysis and synthesis of synthetic peptides, proteins, DNA and RNA oligonucleotides, bioconjugates and other biomolecules.

    You can call us at 1-800-227-0627
    or you can visit our website at
    "www.biosyn.com"

    We are here to help you with all your scientific research needs!

    References

    Bernard et al.A long nuclear-retained non-coding RNA regulates synaptogenesis by modulating gene expression. EMBO J. 2010. 29: 3082-3093. PMID: 20729808.

    Bhartiya et al. Conceptual approaches for lncRNA drug discovery and future strategies. Expert Opin Drug Discov. 2012. 7: 503-513. PMID: 22559214.

    Cabili MN, Trapnell C, Goff L, Koziol M, Tazon-Vega B, et al. (2011); Integrative annotation of human large intergenic noncoding RNAs reveals global properties and specific subclasses. Genes Dev 25: 1915–1927. Access the most recent version at doi:10.1101/gad.17446611.

    Chakalova, L., et al., Replication and transcription: shaping the landscape of the genome. Nat Rev Genet, 2005. 6(9): p. 669-77.

    Gutschner et al. The Noncoding RNA MALAT1 Is a Critical Regulator of the Metastasis Phenotype of Lung Cancer Cells, Cancer Res 2013;73:1180-1189.

    Harrow, J., Frankish, A., Gonzalez, J.M., Tapanari, E., Diekhans, M., Kokocinski, F., Aken, B.L., Barrell, D., Zadissa, A., Searle, S., et al. (2012). GENCODE: the reference human genome annotation for The ENCODE Project. Genome Res. 22, 1760–1774.

    He, Y., et al., The antisense transcriptomes of human cells. Science, 2008. 322(5909): p. 1855-7.

    Jacob, F., and Monod, J. (1961). Genetic regulatory mechanisms in the synthesis of proteins. J. Mol. Biol. 3, 318–356.

    Katayama, S., et al., Antisense transcription in the mammalian transcriptome. Science, 2005. 309(5740): p. 1564-6.

    Khalil, A.M., et al., Many human large intergenic noncoding RNAs associate with chromatin-modifying complexes and affect gene expression. Proc Natl Acad Sci U S A, 2009. 106(28): p. 11667-72.

    Lee et al. RNase H-mediated degradation of toxic RNA in myotonic dystrophy type 1. Proc Natl Acad Sci U S A. 2012. 109(11):4221-6. PMID: 22371589.

    Tong Ihn Lee and Richard A. Young; Transcriptional Regulation and Its Misregulation in Disease. 2013 Cell 152, 1237-1251.

    Ma L, Bajic VB, Zhang Z.; On the classification of long non-coding RNAs. RNA Biol. 2013 Apr 15;10(6), 1-10.

    Ma et al. Molecular Mechanisms and Function Prediction of Long Noncoding RNA, ScientificWorldJournal. 2012; 2012: 541786. PMID: 23319885.

    Mercer, T.R., et al.,Specific expression of long noncoding RNAs in the mouse brain. Proc Natl Acad Sci U S A, 2008. 105(2): p. 716-21.

    Nakaya, H.I., et al., Genome mapping and expression analyses of human intronic noncoding RNAs reveal tissue-specific patterns and enrichment in genes related to regulation of transcription. Genome Biol, 2007. 8(3): p. R43.

    Okada, Y., et al., Comparative expression analysis uncovers novel features of endogenous antisense transcription. Hum Mol Genet, 2008. 17(11): p. 1631-40.

    Sarma et al. Locked nucleic acids (LNAs) reveal sequence requirements and kinetics of Xist RNA localization to the X chromosome. Proc Natl Acad Sci U S A. 2010. 107: 22196-22201. PMID: 21135235.

    Struhl,K., Transcriptional noise and the fidelity of initiation by RNA polymerase II. Nat Struct Mol Biol, 2007. 14(2): p. 103-5.

    Wang KC, Chang HY.; Molecular mechanisms of long noncoding RNAs. Mol Cell. 2011 Sep 16;43(6):904-14. doi: 10.1016/j.molcel.2011.08.018.

    Wilusz et al. Long noncoding RNAs: functional surprises from the RNA world, Genes Dev. 2009. 23: 1494-1504. PMID: 19571179.


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    Solid Phase Polypeptide Synthesis (SPPS)

    The control and regulatory mechanisms for many biological processes are dependent on peptides and proteins derived from α–amino acids. In addition, many modern medicines are now produced from peptides or derivatives of peptides. A few examples are anti-cancer agents, antibiotics or peptide based drugs that control blood pressure. For this reasons α–amino acids and polypeptide chemistry has become a central technology in organic chemistry, biochemistry, biotechnology and medicinal chemistry.

    The automation of polypeptide synthesis and the many improvements made in peptide synthesis instrumentation in recent years have made synthetic peptides and their derivatives more available to the scientific community and the biological industry as a whole. Synthetic peptides can be used to manufacture epitope-specific antibodies, map antibody epitopes and study enzyme binding sites or to design and synthesize novel peptide- or protein-mimetics or even whole enzymes.

    To synthesize a peptide a peptide bond between two amino acids will need to be formed. The exact size of peptides is not well defined but it usually refers to flexible chains of up to 30 to 50 amino acids. However, peptides with up to 100 amino acids within the chain can now be synthesized.

    To synthesize a peptide a peptide bond between two amino acids will need to be formed. The exact size of peptides is not well defined but it usually refers to flexible chains of up to 30 to 50 amino acids. However, peptides with up to 100 amino acids within the chain can now be synthesized.

    The basic concept in solid-phase peptide synthesis is the step-wise construction of a polypeptide chain attached to an insoluble polymeric support (see Figure 1. for general synthesis scheme).

    General Scheme for Solid Phase Peptide Synthesis

    Figure 1: General Scheme for Solid Phase Peptide Synthesis

    This approach permits unreacted reagents to be removed by washing without loss of product. Synthesis of the peptide chain proceeds from the carboxyl end of the amino terminus of the polypeptide.

    The carboxyl moiety of each incoming amino acid is activated by one of several strategies and couples with the α-amino group of the preceding amino acid. The α-amino group of the incoming residue is temporarily blocked in order to prohibit peptide bond formation at this site. The residue is de-blocked at the beginning of the next synthesis cycle. In addition, reactive side chains on the amino acids are modified with appropriate protecting groups. The polypeptide chain is extended by reiteration of the synthesis cycle. Excess reagents are used to drive reactions as close to completion as possible. This generates the maximum possible yield of the final product.

    After fully assembling the peptide the side-chain protecting groups are removed, and the peptide is cleaved from the solid support, using conditions that inflict minimal damage on labile residues. The product is analyzed to verify the sequence thereafter. The synthetic peptide is usually purified by gel chromatography or HPLC. The blocking group used for blocking the α-amino group determines both the synthesis chemistry employed and the nature of the side-chain protecting groups. The two most commonly used α-amino protecting groups are Fmoc (9-fluorenyl-methoxy-carbonyl) and tBoc (tert.-butyloxycarbonyl). Fmoc side-chain protection is provided by ester, ether and urethane derivatives of tert.-butanol, while the corresponding tBoc protecting groups are ester, ether, and urethane derivatives of benzyl alcohol. The latter are usually modified by the introduction of electron-withdrawing halogens for greater acid-stability. Ether and ester derivatives of cyclopentyl or cyclohexyl alcohol are also employed.

    The Fmoc protecting group is base-labile. It is usually removed with a dilute base such as piperidine. The side-chain protecting groups are removed by treatment with trifluoroacetic acid (TFA), which also cleaves the bond anchoring the peptide to the support. The tBoc protecting group is removed with a mild acid (usually dilute TFA). Hydrofluoric acid (HF) can be used both to deprotect the amino acid side chains and to cleave the peptide from the resin support. Fmoc is a gentler method than tBoc since the peptide chain is not subjected to acid at each cycle and has become the major method employed in commercial automated polypeptide synthesis.

    Fmoc Chemistry

    The α-amino groups are protected by the Fmoc (9-fluorenylmethoxycarbonyl) group, while side-chain protection is provided by ester, ether and urethane derivatives of tert.-butanol (see Figure 2. for details).

    Fmoc-Chemistry for Peptide Synthesis

    Figure 2. Fmoc-Chemistry for Peptide Synthesis

    Carboxyl group activation of the incoming amino acid utilizes one of the following methods:

    (1.) BOP/HOBt/NMM:

    The amino acid is mixed with the BOP (Castro's reagent or (benzotriazol-yloxy) tris (dimethylamino) phosphonium hexa-fluoro-phosphate), HOBt (1-hydro-xybenzotriazole) and suff-icient NMM (N-methyl-morpholine) to ionize 50% of the HOBt.

    (2.) DIPCDI:

    DIPCDI (disopropylcarbo-diimide) can be used either with or without HOBt in a solution of DCM:DMF (di-chloromethane and dimethyl-formamide).

    (3.) Active esters:

    These are usually pentafluorophenyl esters (OPfp) of the amino acids. The pentafluorophenyl esters of serine and threonine are oils and therefore are employed in the more convenient form of the dihydrooxobenzotriazine ester (ODhbt).

    tBoc Chemistry

    tBoc (tert-butyl-oxy-carbonyl) chemistry is widely used in peptide synthesis (see Figure 3.).

    tBoc-Chemistry for Peptide Synthesis

    Figure 3. tBoc-Chemistry for Peptide Synthesis

    Side chain protection is done by ester, ether, and urethane derivatives of benzyl alcohol, and modified by the introduction of electron-withdrawing halogens for greater acid-stability. Ether and ester derivatives of cyclopentyl or cyclohexyl alcohol are also employed.

    Activation of the incoming amino acid employs DPCDI in a DCM:DMF solution and may also include HOBt.

    Polypeptide Synthesis Chemistries: A Comparison

    Fmoc tBoc
    Polar polyamide or polysterene supports Polystyrene support
    Fmoc α-amino protection tBoc α-amino protection
    t-Butyl side-chain protection Benzyl side-chain protection
    Mild base deprotection (piperidine) Acid (TFA) deprotection
    No neutralization step Neutralization step required
    Preformed active esters or in situ activation In situ activation required
    TFA cleavage/deprotection HF cleavage/deprotection
    Single solvent system (DMF) Multi-solvent
    Continuous-flow or batch reaction methods Batch reaction method most common
    On-line UV monitoring option Automated monitoring not available
    Range of linkage agents to generate free acids, amides, and fully protected peptides Multiple resins to generate free acids and amides
    Double coupling not recommended Double coupling may be used
    Capping not normally recommended Capping normally recommended
    Fmoc-Protected L-Amino Acid Derivatives

    Fmoc amino acids use usually t-butyl based protection for most labile side-chains. The trityl (Trt) protecting group is recommended for Cys. This is removed when the peptide is cleaved with TFA. The acetamidomethyl (Acm) derivative of Cys is unaffected by TFA treatment, but may be rapidly removed by electrophilic reagents such as mercury or iodine. Oxidative deprotection with iodine yields disulfides directly and is a convenient, clean method of forming intramolecular disulfide links. For Arg side-chain protection, both Mtr and the more easily removed Pmc group are widely used. The amidated side-chains of Gln and Asn are protected either with the standard Tmob or with the more recently available Trt.

    Fmoc-Protected D-Amino Acid Derivatives

    Peptides containing D-amino acids are finding increased use in both research laboratory and pharmaceutical application. Their presence in the peptide allows chain configuration not possible with the L-amino acids alone. Many potent, naturally occurring antibiotics and toxins are cyclic peptides, and often incorporate several D-amino acids to confer a specific conformation as well as make the peptide more resitant to proteolytic attack.


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    Bio-Synthesis provides synthesis of oligonucleotide containing methylated DNA base for epigenetic studies . Epigenetics is the study of changes in heritage control o f gene expression through an identidal DNA sequences , yet maintain different termial phenotypes. This nongenetic cellular memory, which records developmental and environmental cues (and alternative cell states in unicellular organisms), is the basis of epigenetics. The growing interest to fully explain the heritability of complex traits, and to pinpoint the genetic effects in some complex diseases, along with the desire to understand why cells have “deprogramming” into pluripotent/totipotent states, has led epigenetic research into many regulatory systems involving switching a gene from its 'on' to the 'off' state or vise versa in area of DNA methylation, histone modification, nucleosome location, or noncoding RNA. Until 2009, the knowledge about DNA modifications was limited to the introduction of a methyl group at C5 of the cytosine base, which converts 2’-deoxyCytidine (dC) into 5-methyl-2’-deoxyCytidine (mdC).

     methylated dC DNA methylation Epigenetic

     

    This methylation occurs predominantly in CpG islands (areas with high occurrence of the CG motif) of promoters. mdC is introduced by specialized DNA methyltransferase (DNMT) enzymes.

    In 2009, study showed 1,2 the discovery of 5-hydroxymethyl-2’-deoxyCytidine (hmdC), a novel dC modification in Purkinje neurons and embryonic stem cells. Later, a third report found this modification to be strongly enriched in brain tissues associated with higher cognitive functions.3 This new dC modification is generated by the action of a-ketoglutarate dependent TET enzymes (ten eleven translocation), which oxidizes mdC to hmdC. This finding stimulated discussion about active demethylation pathways that could occur, e.g., via base excision repair (BER), with the help of specialized DNA glycosylases. Alternatively, one could envision a process in which the hydroxymethyl group of hmdC is further oxidized to a formyl or carboxyl functionality followed by elimination of either formic acid or carbon dioxide4,5 .

    A number of recent publications provides data that support both pathways. It was discovered that hmdC could be deaminated by activation-induced deaminase (AID) enzymes to provide 5-hydroxymethyl-2’-deoxyUridine (hmdU). This compound was shown to be excised by the SMUG-1 DNA glycosylase6 . After initial failure to detect any further oxidized hmdC derivatives in somatic tissues4, newly developed mass spectrometric technologies, in combination with the available reference compounds, finally enabled researchers to gather strong support for the putative oxidative demethylation pathway. These methods and standards enabled the discovery of 5-formyl-2’-deoxyCytidine (fdC) in differentiating embryonic stem cells.7 Recently, a similar technology also led to the discovery of 5-carboxyl-2’-deoxyCytidine (cdC)8,9, but the amount of fdC and cdC measured differs largely in all three reports.

    Research is currently ongoing to unravel the true levels and fate of these further oxidized dC bases in somatic tissues and in different stem cells. Even along the oxidative pathway, base excision processes have been proposed to play a major role with two reports showing that thymidine DNA glycosylase (TDG) accepts both fdC and cdC as substrates.8,10 A possible oxidative demethylation pathway would clearly rely on the existence of a dedicated decarboxylase that is able to convert cdC back into dC. Such an intriguing decarboxylation would enable nature to remove the 5-methyl group in mdC without introducing DNA strand-breaks that accompany any BER based base removal.

    Bio-Synthesis has has supported epigenetic resarch by providing the synthesis of oligonucletoides containing all the new cytosine analogues: hmdC, fdC and cdC. These modified base can be incorporate during synthesis at any positions using conventional solid-phase oligonucleotide synthesis chemistry but repalcing the standard cytosine DNA base with mehtylated DNA cytosine base.

    • 5-hydroxymethyl-dC [hmdC]
    • 5-hydroxymethyl-dC II [hmdCII]
    • 5-carboxy-dC [cdC]
    • 5-Formyl-dC [fdC]

    The first generation hmdC phosphoramidite was fairly very well accepted but requires fairly harsh synthesis conditions. Therefore, a second generation building block (5-Hydroxymethyl-dC II) developed by Carell and co-workers that is compatible with UltraMild deprotection has been introduced.6 5-Formyl-dC and 5-carboxy-dC may find uses in research into DNA damage and repair processes.

    References:

    1. S. Kriaucionis, and N. Heintz, Science, 2009, 324, 929-30.
    2. M. Tahiliani, et al., Science, 2009, 324, 930-935.
    3. M. Münzel, et al., Angewandte Chemie-International Edition, 2010, 49, 5375-5377.
    4. D. Globisch, et al., PLoS One, 2010, 5, e15367.
    5. S.C. Wu, and Y. Zhang, Nat Rev Mol Cell Biol, 2010, 11, 607-20.
    6. M. Münzel, D. Globisch, C. Trindler, and T. Carell, Org Lett, 2010, 12, 5671-3.
    7. M. Münzel, et al., Improved Synthesis and Evaluation of Oligonucleotides Containing 5-Hydroxymethylcytosine, 5-Formylcytosine and 5-Carboxylcytosine. In Chemistry - A European Journal, 2011, in press.

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    Purification of Synthesized Long RNA Transcripts

    After long RNA synthesis by transcribing from DNA template, unmodified RNA transcripts from standard DNA synthesis are been purified by phenol-chloroform extraction and ethanol precipitation following with spin column chromatography.  For capped RNA syntehsis, non-radioactively labeled RNA or highly specific activity radiolabeled RNA probes, spin column chromatography is the preferred method. When high purity RNA transcript for labeled RAN rpobes for RNase protection assay or foot printing experiments, we recommend gel purificaiton of the transcription product.

    Bio-Synthesis's Long RNA Synthesis transcription service includes phenol-chloroform extraction and ethanol precipitation. Final product are further purified using spin column chromatography. Gel purification is available with an additional fee.

    Characterization of RNA transcription Products

    After transcription from template DNA, RNA transcripts concentration are been determined by measuring with ultraviolet light absorbance at 260 nm wavelength. Evaluation of transcript length, integrity and quantity are further analyzed on an appropriate agarose gel or polyacrylamide gel.

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  • 07/26/13--00:00: Heavy Isotope Labeling
  • Isotopically labeled peptides

    Stable-isotope labeled peptides are used in biomarker discovery and validation as well as in drug and metabolite monitoring, peptide signaling experiments, metabolomics and pharmaco kinetics. Bio-Synthesis, Inc is pleased to supply scientists and researchers with custom synthesized stable-isotope peptides. In contrast to standard amino acids, isotopically labeled amino acids are synthesized by substituting 12C, 14N, and H atoms with 13C, 15N, or Deuterium atoms. These amino acids are non-radioactive and have known molecular weights that are higher than standard amino acids. This molecular weight difference makes stable isotope labeled peptides useful tools for quantitative peptide analysis or protein structure and dynamics determination by mass spectrometry (MS) or nuclear magnetic resonance (NMR) spectroscopy, respectively. When used as surrogate markers for proteins of interest, these peptides are added to allow proteins that they represent to be quantified. The best markers/internal standards are peptides that have exact sequences as the biomarker peptides and carry stable isotopic labels. The isotopes change the mass but not the chemical behavior. Isotope ratio of spiked peptides and peptides obtained from protein biomarkers are measured by mass spectrometry and tandem mass spectrometry to provide absolute quantitation of proteins of interest. Stable-isotope labeled peptide sequences are synthesized using the latest Fmoc solid-phase technology and purified by HPLC. All stable-isotope labeled peptide sequences come with certificate of analysis, mass spectrometric analysis and, stringent analytical HPLC to determine the final purity and assure that you are receiving only the highest quality peptides for absolute quantitation.

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