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Latest Articles of Bio-Synthesis Inc.

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    Abstract:

    Mutations on epidermal growth factor receptor (EGFR) cause a variety of cancers including breast and lung cancers. The single mutation T790M on tyrosine kinase domain of EGFR signifies the response to the popular cancer drug gefitinib, which leads to the development of resistance to gefitinib. Detecting the mutation thus guide effective therapeutical options for patients who are in need of cancer drug treatments. We sought to develop a rapid, reliable detection method for the T790M mutation using bridged nucleic acids (BNA), which has been known to enhance the hybridization affinity of oligonucleotides that contain BNA bases. Oligonucleotides containing BNA bases designed to block PCR reaction against wild-type genes, called BNAclamp, were used to discriminate the presence of mutant genes mixed with a large number of wild-type genes. Real-time PCR in conjugation with BNAclamping allows us to view the different levels of PCR amplifications in the degree of mixture of wild-type and mutant genes. In an effort to explore the possibility, 13-mer long clamps were prepared with various numbers of BNA bases. The clamps containing 9 BNA bases appear to be most effective in blocking the PCR reactions at an optimized concentration to distinguish the mutant from the wild-type genes. In addition to PCR results, the deference in the Tm values for the 7 BNA bases in the 13-mer was the largest among differently designed clamps, which is consistent with the results obtained by real-time PCR. We also examined the degree of sensitivity using the clamp containing 9 BNA bases, revealing that the clamp has the ability to determine the level of mutation as low as 1% mixture oft he mutant and wild-type gene. The effectiveness of blocking the PCR reaction with only 13-mers containing BNA bases allowed us to detect a single mutation, and thus, this BNAclamping real time PCR technology may offer a promising, new avenue to detect clinically importantmutations in the future.

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     PCR protocol for BNA clamping

    Many mutations in human signaling and membrane receptor proteins are the cause of many cancers. Screening for these mutations allows identification of biomarkers that indicate the risk of a patient’s for developing cancer. In addition, after a particular cancer type in a patient has been diagnosed, it is important to screen for resistant of the identified cancer type to therapeutic drugs. For example, the single T790M mutation in the tyrosine kinase domain of the epidermal growth factor receptor (EGRF) allows cancers carrying this mutation to be resistant to gefitinib. Gefitinib is a therapeutic cancer drug used to treat certain breast and lung cancers. Gefitinib inhibits EGRF and interrupts cellular signaling through EGRF in target cells. The cell surface transmembrane glycoprotein EGRF binds to epidermal growth factor. In doing so, it induces receptor dimerization and tyrosine autophosphorylation that leads to cell proliferation. Mutations in this gene are known to be present in lung cancer. Lung cancer is a malignancy that affects lung tissues. The most common forms are divided into three major subtypes: squamous cell carcinoma, adenocarcinoma, and large cell lung cancer.

    Another example is KRAS or ViKi-ras2 Kirsten rat sarcoma viral oncogene homolog. The KRAS gene is coding for a protein called K-Ras primarily involved in cell division. KRAS is part of the RAS/MAPK pathway in which it relays signals from outside the cell to the cell’s nucleus. The K-Ras protein is an oncogene and a GTPase that acts as a switch that is turned on and off by GTP and GDP molecules. Several mutations in this gene have been identified in people with disorders, and some of them are acquired during a human’s lifetime. Each mutation changes a single amino acid in the protein. Some of these mutations alter chemical signaling in cells throughout the body and can interfere with normal development of many organs or tissues. Mutated oncogenes have the potential to cause cells to become cancerous.

    A newly developed bridged-nucleic acid (BNA) based PCR-clamping method enables detection of single point mutations such as the T790M mutation in the tyrosine kinase domain of EGRF in a quite simple and cost-effective manner. The BNA clamp selectively suppresses the amplification of the perfect match (wild type) but does not inhibit the amplification of a sequence that differs by as little as one base, for example, mutations. The technique is called PCR Clamping and allows resolving single base differences present in template strands to amplify and detect. This BNA based method allows the selective amplification of many DNA target sequences that differ by a single base pair. The high affinity and specificity of the BNA probe for its complementary nucleic acid sequence prevent the target sequence to function as a primer.

    Bridged nucleic acids (BNAs) are known to enhance the hybridization affinity of oligonucleotides that contain BNA bases. BNAclamps are designed to block PCR reaction against wild-type genes to discriminate the presence of mutant genes mixed in with a large number of wild-type genes. The use of Real-Time PCR (RT-PCR) together with BNAclamping allows the detection of different levels of PCR amplifications in mixtures of wild-type and mutant genes.

    The polymerase chain reaction, or PCR, is a powerful laboratory technique that allows for the amplification of specific DNA sequences. Since its discovery in 1985, many variations of this method have been introduced and carried out. Today, PCR has become an indispensable part of modern molecular cloning techniques. With the help of PCR, a defined target sequence can be readily and selectively amplified in a quasi-exponential chain reaction. PCR enables the generation of millions of copies, and the method has also been adapted to a wide variety of tasks.  PCR is now used in many molecular biology techniques such as DNA sequencing, in vitro mutagenesis, and mutation detection, cloning of cDNA and genomic DNA, as well as allotyping.

    However, to get started with any PCR experiment a basic protocol is always a good starting point. Depending on the target DNA modifications to the protocol, may be needed. Because PCR is used for so many applications it is impossible to describe a single set of conditions that will guarantee success in all situations or without further optimizing the protocol for the specific application.

    1.    Selection of target substrates and PCR primers

    The nature of the target DNA together with the specific experiment dictates the nature of the protocol used. The desired substrate DNA should be chosen as clean as possible as well as uncontaminated with other DNA. Select the PCR primers needed.

    2.    Setting up the reaction

    Setting up a PCR requires the following components:

    • A thermo stable DNA polymerase to catalyze template-dependent synthesis of DNA.

    • A pair of synthetic oligonucleotides to prime DNA synthesis. A forward and reverse primer is needed.

    • Deoxynucleoside triphosphate (dNTPs). A standard reaction requires equimolar amounts of dATP, aTTP, dCTP, and dGTP. Recommended concentrations for Taq polymerase are 200 to 250 µM plus 1.5 mM MgCl2 in reaction mixures.

    • Divalent cations. Mg, Mn.

       
    • Buffer to maintain pH.

    • Monovalent cations. KCl. A standard PCR buffer contains 50 mM KCl. For the amplification of shorter DNA fragments concentrations of ~70 to 100 mM KCl may work better. Circular DNA is amplified slightly less effciently than linear DNAs.

    • Template DNA. Target DNA, single- or double-stranded.

    In principle PCR can detect a single target molecule, however, in practice several thousand copies of the target DNA are seeded into the reaction mixture.

    3.   Basic BNA PCR protocol


    1. Dissolve BNA oligonucleotides in PCR water to a final solution of 100 µM.

    2. Prepare a stock solution, for example by dissolving 25.44 nmole BNA oligonucleotides in 254.4 µL water.

    3. Next, dilute 10 µL stock solution in 90 µL PCR water. Use this as a working solution (10 µM).

    4. Start the PCR using the following protocol:

    4.    Example:  BNA clamping experiment

     

    Reagent

    Aliquot

    1

    2 x SYBR green Mix

     5 µL

    2

    10 µM Forward primer

    0.2 µL (This can be adjusted according to experiment)

    3

    10 µM Reverse primer

    0.2 µL (This can be adjusted according to experiment)

    4

    Target sample (~ 104 copy number or greater)

    1 µL

    5

    10 µM BNA clamp

    1.4 µL (This can be adjusted as needed)

    6

    H2O

    2.2 µL

     

    Total PCR reaction volume

    10 µL

              

    5.   Real-Time PCR conditions    

    1 cycle:  95 oC, 10 min; 50 cycles:  95 oC, 15 sec; 60 oC, for 1 min.

    This means an initial denaturating step of 10 minutes at 95 °C is followed by 50 cycles of 95 °C for 15 seconds and 60 °C for 1 minute.

    Reference

    Molecular Cloning: A laboratory manual. 4th edition. Green and Sambrook. Cold Spring Harbor Laboratory Press. 2012.

     


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    The Polymerase Chain Reaction or PCR


    The Polymerase Chain Reaction (PCR) is a technique that allows making many copies of a piece of DNA in a laboratory using readily available reagents. During the reaction, the number of copies increases exponentially. Therefore, within a few hours more than 100 billion copies of a DNA piece can be made. The availability of DNA polymerases and synthetic oligo-nucleotides made this technique possible. PCR has now become an alternative to cloning since it allows amplifying specific sequences in a complex mixture. In addition, PCR combined with sequencing techniques allows identification of mutant alleles in a DNA sample specifically, rapidly and with high sensitivity.


    The PCR technology allows specifically amplifying a target DNA sequence from a tiny amount of starting material. In earlier versions of PCR the Klenow fragment of E. coli DNA polymerase, I was used. However, the Klenow fragment is not
    thermo-stable. The introduction of a thermo-stable DNA polymerase, such as the DNA polymerase found in Thermus aquaticus, resulted in a major technological breakthrough in the development of PCR methodologies.

    PCR can be categorized into different groups or methods based on how the techniques are used. However, all these methods follow the same basic steps and principal. 

     

    Figure 1: Models of TAQ polymerase. Three different depictions of the same model of TAQ polymerase are illustrated (Eom et al. 1996). 

    Eoam, Wang, and Steitz solved the co-crystal structure of Taq polymerase with a blunt-ended duplex DNA bound to the polymerase active-site cleft. The structure indicates that the DNA neither bends nor goes through the large polymerase cleft. The structural conformation of the bound DNA is between the B and A forms. The model of the structure showed that a wide minor groove allows access to protein side chains. The side chains are hydrogen-bonded to the N3 of purines and the O2 of pyrimidines at the blunt-end terminus. 

    DNA polymerases catalyze the synthesis of long polynucleotide chains. The synthesis starts from monomeric deoxynucleotide triphosphates in the presence of an original parental DNA strand. The DNA strand serves as a template for the synthesis of a new complementary DNA strand. The synthesis proceeds in the 5’ to 3’ direction. The polymerization occurs from the 5’ α-phosphate of the deoxynucleoside triphosphate to the 3’ terminal hydroxyl group of the growing DNA strand. DNA polymerases require a short segment of DNA to anneal to a complementary sequence. This DNA sequence or oligonucleotide is called a primer since it is needed to prime the synthesis. 

    The Polymerase Chain Reaction or PCR was discovered, conceived or invented by Kary B. Mullis in 1983. According to him he stumbled upon this reaction when driving in northern California during a moonlit night. Furthermore, as pointed out by him, the reaction allows generating up to 100 billion similar DNA molecules from a single DNA molecule within a few hours. The reaction can be performed in a test tube but also requires a few reagents and a source of heat. The introduction of the PCR reaction has made life much easier for molecular biologists, allowing them to produce as much DNA as they want. The technology has spread throughout the biological sciences with tremendous speed. However, since the reaction involves thermal cycling the most important piece of the ultimately improved PCR technology turned out to be the use of a heat stable DNA polymerase. The polymerase that was originally extracted from the bacterium Thermus aquaticus is now used in almost all PCR reactions. Thermus aquaticus lives in hot springs. The polymerase chain reaction has become the ultimate game-changing technology in molecular biology. In 1993 Kary B. Mullis was awarded the Nobel Prize in Chemistry “for contributions to the developments of methods within DNA-based chemistry” jointly with Michael Smith. Kary B. Mullis received one-half of the prize for his invention of the PCR method. Michael Smith received the other half for his contributions to oligonucleotide-based, site-directed mutagenesis and development for protein studies. {http://www.nobelprize.org/nobel_prizes/chemistry/laureates/1993/}.

    Dr. Mullis now has his own website: http://www.karymullis.com/pcr.shtml.


    Features of the polymerase chain reaction

    • The Polymerase Chain Reaction (PCR) selectively amplifies a target DNA molecule.
    • It allows the extension of short single-stranded synthetic oligonucleotides, primers, during repeated cycles of heat denaturation, primer annealing, and primer extension. 
    • PCR is a cyclic process in which a sequence of steps is repeated over and over again. 
    • Double-stranded fragments of DNA are separated into single strands by mild heating called denaturation. 
    • A short, synthetic oligonucleotides primer is added to the reaction mixture, along with the four deoxyribonucleotide triphosphates dATP, dGTP, dCTP, dTTP, and a DNA polymerase now usually a Taq DNA polymerase.
    • DNA polymerase catalyzes the synthesis of complementary new strands.


    Reference

    Eom SH, Wang J, Steitz TA.; Structure of Taq polymerase with DNA at the polymerase active site. Nature. 1996 Jul 18; 382(6588):278-81.

    Faloona, F., Weiss, S., Ferre, F., and Mullis, K. 1990. Direct detection of HIV sequences in blood high-gain polymerase chain reaction [abstract]. In: 6th International Conference on AIDS, University of California, San Francisco: San Francisco (CA). Abstract 1019.

    Gelfand, D.H. and White, T.J. 1990. Thermostable DNA polymerases. In: PCR Protocols: A Guide to Methods and Applications. Innis, M.A., Gelfand, D.H., Sninsky, J.J., and White, T.J., eds. San Diego: Academic Press. 129–141.

    Holland, P.M., Abramson, R.D., Watson, R., and Gelfand, D.H. 1991. Detection of specific polymerase chain reaction product by utilizing the 5´→3´ exonuclease activity of Thermus aquaticus DNA polymerase. Proc. Natl. Acad. Sci. USA 88:7276–7280.

    Innis, M.A., Myambo, K.B., Gelfand, D.H., and Brow, M.A. 1988. DNA sequencing with Thermusaquaticus DNA polymerase and direct sequencing of polymerase chain reaction-amplified  DNA. Proc. Natl. Acad. Sci. USA 85:9436–9440.

    Mullis K.  In Methods In Enzymology, Vol.155, 335, 1987.

    Kary B. Mullis; The Unusual Origin of the Polymerase Chain Reaction. SCIENTIFIC AMERICAN April 1990. 56-65.



     




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     N-acetylgalactosamine


    N-acetylgalactosamine, 
    or n-acetyl-α-D-galactosamine, or n-acetyl-α-D-galactosamine, alpha-GalNAc; TN saccharide; alpha-GalpNAc; GalNAc-alpha; n-acetyl- α-D-galactosamine; or N-acetyl-alpha-D-galactosamine, is an amino sugar derivative of galactose. In humans, it is the terminal carbohydrate of the blood group A antigen.




    Figure 1: Molecular models of N-acetylgalactosamine or GalNAc.



    Glycosylation is a common post-translational covalent modification found on specific amino acid residues in glycoproteins.Glycosylation refers to the enzymatic process of attaching oligosaccharides to proteins to form glycoproteins or glycosylated proteins or glycans. For example, O-N-acetylgalactosamine (O-GalNAc) moieties are conjugated to the hydroxy oxygen of serine and threonine side chains in O-linked glycans. In particular, O-glycosylation, is a common covalent modification of serine (S, Ser) and threonine (T, Thr) present in glycoproteins such as mucins.

    Mucins are highly glycosylated proteins that form a physical barrier in epithelial cells. Epithelial cells are membranous cellular tissue cells that cover free surfaces or lines, tubes or cavities of animal bodies. Epithelial cells serve to enclose and protect other parts of the body, to produce secretions and excretions, and to function in cell or tissue assimilation.Transmembrane mucins are also known to contribute to the physical barrier and to transmit growth and survival signals to the interior of cells. In many epithelial surfaces, mucins shield these surfaces against physical and chemical damage and also protect the cells from infections by pathogens. O-glycans in mucins begin with an α-linkedN-acetylgalactosamine residue linked to a serine or threonine residue. The N-acetylgalactosamine residue is extended with sugars such as galactose, N-acetylgalactosamine, fucose, or sialic acid. However, mannose, glucose, or xylose residues appear to not being used. There are several O-GalNAc glycan core structures, and other mucin O-glycans are often branched as well. Many sugar structures on mucins are antigens. O-Glycan structures for mucins and blood groups are listed in Table 1. A variety of chemical, enzymatic, and spectroscopic methods are used for the analysis of these sugar linkages and structures on mucin glycans. Glycopeptides containing O-glycan moities can be routinely synthesized using automated solid phase peptide synthesis methods.  http://www.biosyn.com/faq/custom-glycopeptide-synthesis.aspx


    Table 1:  Structures of O-glycan cores and antigenic epitopes found in mucins

    O-Glycan

    Structure

    Core

     

    Tn antigen

    GalNAcαSer/Thr

    Sialyl-Tn antigen

    Siaα2-6GalNAcαSer/Thr

    Core 1 or T antigen

    Galβ1-3GalNAcαSer/Thr

    Core 2

    GlcNAcβ1-6(Galβ1-3)GalNAcαSer/Thr

    Core 3

    GlcNAcβ1-3GalNAcαSer/Thr

    Core 4

    GlcNAcβ1-6(GlcNAcβ1-3)GalNAcαSer/Thr

    Core 5

    GalNAcα1-3GalNAcαSer/Thr

    Core 6

    GlcNAcβ1-6GalNAcαSer/Thr

    Core 7

    GalNAcα1-6GalNAcαSer/Thr

    Core 8

    Galα1-3GalNAcαSer/Thr

     

     

    Epitopes

     

    Blood groups O, H

    Fucα1-2Gal-

    Blood group A

    GalNAcα1-3(Fucα1-2)Gal-

    Blood group B

    Galα1-3(Fucα1-2)Gal-

    Linear B

    Galα1-3Gal-

    Blood group i

    Galβ1-4GlcNAcβ1-3Gal-

    Blood group I

    Galβ1-4GlcNAcβ1-6(Galβ1-4GlcNAcβ1-3)Gal-

    Blood group Sd(a), Cad

    GalNAcβ1-4(Siaα2-3)Gal-

    Blood group Lewisa

    Galβ1-3(Fucα1-4)GlcNAc-

    Blood group Lewisx

    Galβ1-4(Fucα1-3)GlcNAc-

    Blood group sialyl-Lewisx

    Siaα2-3Galβ1-4(Fucα1-3)GlcNAc-

    Blood group Lewisy

    Fucα1-2Galβ1-4(Fucα1-3)GlcNAc-

     

    α-N-acetylgalactosaminidase

    The enzyme α-N-acetylgalactosaminidase (a-NAGAL, E.C. 3.2.1.49), a lysosomal exoglycosidase, cleaves terminal a-N-acetylgalactosamine residues from glycopeptides and glycolipids and removes them primarily from serine and threonine residues. In humans, a deficiency of a-NAGAL activity results in the lysosomal storage disorders Schindler and Kanzaki diseases. Clark and Garman determined the structure of this enzyme in 2009. Loss of enzyme activity or its lower activity leads to accumulation of glycolipids and glycopeptides in tissues. This accumulation ultimately leads to clinical symptoms such as the ones observed in Schindler disease, a neurodegenerative disorder. Models of the structure of α-N-acetylgalactosaminidase are shown in the following figure at 1.9 A resolution.




    Figure 2: Molecular models of α-N-acetylgalactosaminidase (α-NAGAL, EC. 3.2.1.49). A lysosomal exoglycosidase that cleaves terminal α-N-acetylgalactosamine residues from glycopeptides and glycolipids. In humans, a 

    deficiency of α-NAGAL activity results in the lysosomal storage disorders Schindler and Kanzaki diseases. The reaction catalyzed by the enzyme is shown in the right upper right. Molecular models for GalNAc are shown to the right. Models of the enzyme structure are shown on the left and in the upper middle panel. Ribbon diagrams of the human α-NAGAL dimer and monomer with the enzymatic product α-GalNAc in the active sites are shown (Clark and Garman, 2009).


    Functions of O-GalNAc glycans


    O-GalNAc glycosylation is a post-translational modification. O-GalNAc glycosylation appears to be an essential process since all mammalian cell types studied so far express 
    polypeptide N-acetylgalactosamine (GalNAc) transferases (ppGalNAcTs). The enzyme polypeptide N-acetylgalactosamine (GalNAc) transferase (ppGalNAcT) when active initiates the covalent linkage of GalNAc to serine and threonine residues of proteins. Many ppGalNAcTs operate within multicellular organisms. However, they differ in expression patterns and substrate selectivity. It appears that ppGalNAcTs are important for differentially modulated regulatory processes in animal development, physiology, and possibly in disease. For example, animals lacking ppGalNAcT-1 are markedly impaired in immunoglobulin G production, have increased germinal center B-cell apoptosis and reduced levels of plasma B cells.


    O-glycans are hydrophilic and usually negatively charged. These characteristics allow them to bind water and salts making them major contributors to the viscosity and adhesiveness of mucus. Removal of microbes and particles trapped in mucus is an important physiological process. O-GalNAC glycans significantly influence the conformation of the attached protein. O-glycosylation of mucins provides almost complete protection from protease degradation. Similarly, this can be the case for other glycoproteins as well. Furthermore, it is thought that O-glycans of cell-surface receptors may regulate receptor stability and expression levels. Also, selectin-glycan interactions are important for the attachment of leukocytes to the capillary endothelium during homing of lymphocytes or the extravasation of leukocytes during the inflammatory response. To exemplify this, the removal of core 2 O-GalNAc glycans from mice by eliminating the C2GnT-1 gene resulted in a sever deficiency in the immune system of these mice. Cancer cells that often express sialyl LewisX epitopes appear to use the selectin-binding properties of the glycan to invade other cell tissue. In cancer, it was observed that the biosynthesis of O-GalNAc glycans is often abnormal, which may affect the biology and survival of the cancer cell. Finally, O-glycosylated glycoproteins may be important during reproduction and fertilization.

    References


    Nathaniel E. Clark and Scott C. Garman; The 1.9 Å structure of human α-N-acetylgalactosaminidase: The molecular basis of Schindler and Kanzaki diseases. J Mol Biol. 2009 October 23; 393(2): 435–447. doi:10.1016/j.jmb.2009.08.021.


    Delta masses: http://www.ionsource.com/; https://www.abrf.org/index.cfm/dm.home?AvgMass=all

    Donald M. Marcus, Elvin A. Kabat, Gerald Schiffman (1964). "Immunochemical Studies on Blood Groups. XXXI. Destruction of Blood Group A Activity by an Enzyme from Clostridium tertium Which Deacetylates N-Acetylgalactosamine in Intact Blood Group Substances". Biochemistry3: 437–443. doi:10.1021/bi00891a023

    Tenno M, Ohtsubo K, Hagen FK, et al. Initiation of Protein O Glycosylation by the Polypeptide GalNAcT-1 in Vascular Biology and Humoral Immunity . Molecular and Cellular Biology. 2007;27(24):8783-8796. doi:10.1128/MCB.01204-07. http://www.ncbi.nlm.nih.gov/pubmed/17923703


    NIH Book

    Essentials of Glycobiology. 2nd edition. http://www.ncbi.nlm.nih.gov/books/NBK1896/ . Essentials of Glycobiology, 2nd edition. Editors: Ajit Varki, Richard D Cummings, Jeffrey D Esko, Hudson H Freeze, Pamela Stanley, Carolyn R Bertozzi, Gerald W Hart, and Marilynn E Etzler. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 2009. ISBN-13: 9780879697709.



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    During aging and disease, cellular damage is accumulated in cells of organisms such as animals and humans. Interstrand DNA crosslinks (ICLs) are formed by metabolic products as well as chemotherapeutic reagents (Muniandy et al. 2010).  The maintenance of the genomic integrity of cells is crucial for the survival of all organisms (Ratanaphan, 2012). Damage in the genomic DNA causes disorders, aging, and cancer. A variety of endogenous metabolites, exposure to environmental agents and cancer chemotherapeutics having two reactive groups are known to cause lesions in DNA by covalently joining two DNA strands together. The resulting cross-links are called interstrand cross-links that prevent the separation of the two joined DNA strands. Several synthetic DNA interstrand cross-links have been used for the elucidation of repair pathways (Guainazzi and Scharer, 2010). Synthetic control ICLs are valuable tools for the study of DNA repair pathways for the identification of new therapeutic targets for a more selective cancer chemotherapy.


    Figure 1: Structure of ethylene ICL DNA (O’Flaherty et al. 2014). The ethylene ICL is highlighted as atomic spheres in the model to the right. This lethal ICL is formed by the
    bisalkylating agent 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). BCNU is used in cancer chemotherapy to hinder cellular proliferation. The ethylene linkage ICL connecting the two DNA strands at the N1 atom of 2'-deoxyguanosine and N3 atom of 2'-deoxycytidine is generated by BCNU.


    Essential cellular processes including DNA replication and transcription are inhibited by these cross-links. However, it appears to be the job of stem cells to replenish needed tissue cells. It is thought that the damage stem cells sustain over time will compromise their ability to maintain healthy tissues. Information now available about ICLs has increased dramatically during the last decade. For example, Clauson et al. in 2013 reported endogenous sources for DNA ICLs as well as methods describing how to detect, identify and analyze endogenous lesions and repair factors. Table 1 lists common endogenous sources that cause ICLs. Table 2 contains a list of methods useful for the detection and measurement of ICL lesions and their repair.


    Table 1: Endogenous sources of DNA interstrand crosslinks.

     

    ICL-inducing compounds

    Target in DNA

    Synthetic model

    Endogenous sources

    Aldehydes: Trans-4-

    hydroxynonenal, acetaldehyde,

    malondialdehyde, acrolein,

    formaldehyde, crotonaldehyde

    5-GC – nondistorting

    5-CG – distorting

     

    Stabilized trimethylene

    ICL between two N2-G

     

    Lipid peroxidation;

    metabolism of dietary

    components including:

    coffee, ripe fruit and

    alcohol

    Nitric oxide, nitrous acid

    5-GC < 5-CG –

    distorting

     

    Nitrous acid induced

    ICL two N2-G

     

    Cell signaling;

    acidification of dietary

    nitrates

    Oxidized abasic lesion: 5-(2-

    phosphoryl- 1,4,dioxobutane)

    A on the opposite

    strand, 3to the abasic site

    Photolabile precursor built into a ss oligonucleotide

    Hypoxic conditions

    Ring-open aldehyde form of an abasic site 3to a C residue

    G opposite the C

     

    AP site built into a ss oligonucleotide; ICL stabilized by reduction with NaCNBH3

    Spontaneous hydrolysis of

    purines or BER repair

    intermediates

     

     

    Table 2: Methods to detect and measure ICL lesions and their repair.

     

    Method

    Endpoint Measured

    Advantages

    Disadvantages

    Local damage with psoralen + UV-A

    Covalent addition of psoralen to chromatin in aportion of a cell nucleus

    Used to identify proteins that co-localize with ICLs to determine the order of events during ICL repair

    Can only be used in cells cultured in monolayers;

    Cannot prove the lesions are ICLs

     

    Mass spectrometry

     

    Levels of mitomycin C or other specific ICL lesions in genomic DNA

    Highly specific if use

    tandem MS; Highly sensitive if used

    isotopically labeled

    internal standard;

    Applicable to cells,

    tissues or body fluids

    Limited to a single lesion per analysis and those for which synthetic standards

    are available

     

    Alkaline COMET assay

    DNA damage that restricts the electrophoretic mobility of DNA

     

    Single-cell measurement of DNA

    damage; Sensitive

     

    Subjective quantification;

    Cannot be used on lesions that are unstable in alkali; Cannot be used on tissues; Genome contains multiple types of DNA damage

    Note: Both tables are from Clauson et al. (2013).


    Recently, scientists presented data that support the idea that stem cells may have intrinsic protective mechanisms that keep them healthy (Katajisto et al. 2015). These recent findings showed that mechanisms exist for mammalianstem like cells to asymmetrically sort aged and young mitochondria. These properties appear to be important to maintain stemness. Stemness is defined as unique characteristics of stem cells which distinguish these cells from normal cells. Stem cells are progenitor or ancestor cells that are able to self-renew and maintain other cells types present in organs. The ability of embryonic (ESCs) and adult tissue stem cells to self-renew and develop into multiple cell lineages is at the heart of the basic definition of “stemness”. Normally, this discovered self-renewal machinery is restricted to stem cells. Therefore one cancer theory suggests that the aberrant activation of the self-renewal machinery in normal cells may be responsible for the initiation of cancer.



    Many chemicals are known to produce DNA interstrand cross-links. However, our understanding of the repair of these interstrand cross-links is still incomplete. Research that aims to better understand repair mechanisms for these cross-links is relevant to cancer chemotherapy since tumors often develop resistance to bifunctional anticancer drugs (Noll et al. 2006). Bifunctional anticancer drugs are used in tumor chemotherapy because the tumor tissue growths faster than normal tissue cells and are therefore sensitive to the therapy.



    Chemicals that are known to induce interstrand crosslinks are potent genotoxic agents such as bifunctional alkylating agents capable of forming ICLs (Vogel, et al. 1998). Chemotherapy drugs that form ICLs include psoralen and bifunctional alkylating agents such as mechlorethamine, chlorambucil, mitomycin C, and cisplatin. Psoralen induces Thymidine-Thymidine interstrand cross-links
    (T:T ICLs) induced by irradiation with long-wavelength UV light. However, other compounds react with the nitrogen 7 (N7) on the guanine moiety to form guanidine-guanidine (G:G) ICLs.



    ICLs caused by nitrogen mustard are illustrated in figure 2A. Lipid peroxidation products such as acrolein, crotonaldehyde and malondialdehyde which are bifunctional alkylating agents have also been reported to cause ICLs. The cross-links occur at the guanine base. Figure 2B shows the general structure for synthetic ICLs as reported by Guainazzi and Schärer in 2010 and Mukherjee et al. in 2014.


    Figure 2. (A) ICLs formed by nitrogen mustard; (B) ICL analogues prepared by Mukherjee et al. 2014 (The Schärer research group).



    DNA inter-strand cross-links (ICLs) are toxic DNA lesions and prevent the unwinding of DNA duplex by helicases and thus blocks DNA replication and/or DNA transcription. ICL represent severe DNA lesions, which are highly cytotoxic and genotoxic, and if left intact, can lead to cell death. The repair of ICL lesion has become a hot research topic in the past decade. Unfortunately, the repair mechanisms involved are still not well understood and many biochemical steps involved are still unknown.  Nucleotide excision repair (NER) pathways, first discovered in E. coli, are involved in the repair of UV-radiation-induced DNA damage. The base excision repair (BER) pathway and homologous recombination serve to eliminate deleterious
    lesion from chromosomes and participate in the repair process (Noll et al. 2006).  Raeschle et al. in 2008 suggested that nucleotide extension requires DNA polymerase ζ.  Furthermore, they argue that a significant portion of the input DNA is fully repaired, but not if DNA replication is blocked. Experiments performed by this research group helped to establish a mechanism for ICL repair that revealed how the repair process is coupled to DNA replication.



    A most recent plausible mechanism involves the Fanconi anemia pathway (Deans and West; 2011). Fanconi anemia (FA) is a disorder that is associated with a failure in DNA repair.  FA is a genetically heterogeneous recessive disorder characterized by cytogenetic instability, hypersensitivity to DNA
    cross-linking agents, increased chromosomal breakage, and defective DNA repair. The disorder leads to progressive bone marrow failure and predisposition to cancer. Members of the Fanconi anemia complementation group do not share sequence similarity but are related by how they assembly into a common nuclear protein complex. The gene encodes the protein for complementation group M. Sixteen different genes are known to be involved in Fanconi anemia. The gene products participate in the repair of DNA interstrand cross-links as well as other lesions that block replication fork progression (Coulthard et al. 2013). Eight of the FA proteins assemble in the FA core complex. The protein complex associates with chromatin which leads to the mono-ubiquitination of FANCD2 and FANCI (FANCA-FANCQ).  The Fanconi anemia complementation group (FANC) currently includes the proteins FANCA, FANCB, FANCC, FANCD1 (also called BRCA2), FANCD2, FANCE, FANCF, FANCG, FANCI, FANCJ (also called BRIP1), FANCL, FANCM and FANCN (also called PALB2).  Bogliolo et al. in 2013 reported that mutations in ERCC4, encoding the DNA-repair endonucleose XPF, cause Fanconi Anemia. Biochemical and functional analysis demonstrated that the identified FA-causing ERCC4 mutations strongly disrupt the function of XPF in DNA ICL repair without severely compromising nucleotide excision repair. Whole-exome sequence analysis of blood DNA from one patient revealed a missense mutation in exon 11 (c.2065C > A [p. Arg689Ser]) and a 5 bp deletion in exon 8 leading to a frameshift and premature termination of translation (c.1484_488delCTCAA [p. Thr495Asnfs*6]).  Furthermore, sanger sequence analysis of blood DNA from another patient revealed a missense mutation in exon 4 (c.689T > C [p.Leu230Pro]) and a 28 bp duplication in exon 11 leading to a frameshift and a premature stop codon (c.2371_2398dup28 [p.Ile800Thrfs*24]).

    The
    FANCM protein (defective in FA complementation group M) and the FAAP24 protein complex target other FA proteins to sites of DNA damage. The structure model of the architecture of the DNA recognition elements of the Fanconi anemia FANCM-FAAp24 complex is shown in the following illustration (Figure 3).

    Figure 3: The model of the crystal structure of an S-Shaped FANCMCTD-FAAP24 Complex Bound to dsDNA is depicted (Coulthard et al, 2013).


    Model for the ICL Repair Mechanism


    Raeschle et al. in 2008 p
    roposed a model for the ICL Repair Mechanism involving the Fanconi anemia pathway based on their experimental data gained from a cell free system base on Xenopus Egg Extracts that support ICL repair. The research group found that during DNA replication of a plasmid containing a site-specific ICL, two replication forks converge on the cross-link. The bypass of the lesions involves the advance of a nascent leading strand to within one nucleotide of the cross-link. Incisions and translesion DNA synthesis followed next. Finally, extension of the nascent strand beyond the lesion occurs. The experimental results suggested that the extension requires DNA polymerase ζ. Unless DNA replication is blocked a significant portion of the cross-linked DNA is fully repaired. The following shows the individual proposed steps involved in the repair mechanism.



    A. Formation of Interstrand Cross-link (ICL)
    : A DNA strand containing a nitrogen mustard-like ICL is illustrated in the following figure.

    B. Fork Pausing: When the DNA strands are replicated, the leading strands of two converging replication forks initially stall around 20–40 nucleotides (nt) from the lesion. This is called “fork pausing”. 


    C. Extension to the ICL and possibly replisome remodeling: Next, one leading strand (in red) is then extended to within 1 nt of the ICL (see next figure below). This step is thought to require replisome remodeling. The replisome complex carries out DNA replication starting at the replication origin. This protein complex forms the replication fork and contains several enzymes, including a helicase, a primase, and a DNA polymerase. The replication fork duplicates both DNA strands, the leading and the lagging strand. See the review from Lindås& Bernander (in 2013) for more details.


    D. In Chromatid uncoupling by incision: In the next step the two sister chromatids that are joined via the ICL are uncoupled via dual incisions on either side of the ICL. The endonucleases that are thought to be involved in the repair process are the endonucleases XPF and Mus81.


    E.  Nucleotide insertion: Next, a translesion DNA polymerase, possibly Rev1, inserts a nucleotide across from the adducted base.


    F.  Extension past the ICL: In the following step, a DNA polymerase ζ extends the nascent strand beyond the ICL. (Makarova and Burgers, 2015). The DNA polymerase ζ is known to be involved in DNA replication and repair.


    G. Repaired DNA Duplexes are generated: Finally, two fully repaired DNA duplexes are generated through the action of nucleotide excision repair (NER) on the top duplex and homologous recombination (HR) on the bottom duplex.

     

    The restriction enzyme Accused for the analysis of the repair product recognizes the sequence GT(A,C)(T,G)*A*C and generates fragments with 5'-cohesive termini.

    However, is has been noted that repair is quite slow. It can take several hours for the repair reaction to complete, in vitro and in mammalian cells.

    One of the challenges related to ICL studies at the molecular level is the preparation of DNA duplex substrates that have a single cross-link at a defined site in the duplex. Synthetic ICLs with cross-linked bases site-specifically incorporated into the duplex provides good models for DNA lesion repair and transcription studies as well as mechanism studies of some chemotherapy drugs. However, the preparation of ICLs is difficult because normally two modified bases should be incorporated into the oligonucleotide sequences, and post-synthetic modification is required. A good example is the method published recently by the Schärer group to make ICL mimics that are formed by nitrogen mustard (Figure 1B).

    -.-

    Bio-synthesis Inc. has over 30 years of experience in oligonucleotide synthesis and offers comprehensive services for customized oligonucleotide synthesis, modification, labeling and conjugation, including the synthesis of inter-strand crosslink analogues upon request.



    Reference

    Bogliolo M, Schuster B, Stoepker C, Derkunt B, Su Y, Raams A, Trujillo JP, Minguillón J, Ramírez MJ, Pujol R, Casado JA, Baños R, Rio P, Knies K, Zúñiga S, Benítez J, Bueren JA, Jaspers NG, Schärer OD, de Winter JP, Schindler D, Surrallés J. Mutations in ERCC4, encoding the DNA-repair endonuclease XPF, cause Fanconi anemia. Am J Hum Genet. 2013 May 2;92(5):800-6. doi: 10.1016/j.ajhg.2013.04.002. Epub 2013 Apr 25.


    Cheryl Clauson, Orlando D. Schärer, Laura Niedernhofer;  Advances in understanding the complex mechanisms of DNA interstrand crosslink repair. Cold Spring Harb Perspect Med. Author manuscript; available in PMC 2014 August 6. Published in final edited form as: Cold Spring Harb Perspect Med. 2013 October; 3(10): a012732.  PMCID: PMC4123742.

    Coulthard R, Deans AJ, Swuec P, Bowles M, Costa A, West SC, McDonald NQ. Architecture and DNA recognition elements of the Fanconi anemia FANCM-FAAP24 complex. Structure. 2013 Sep 3;21(9):1648-58. doi: 10.1016/j.str.2013.07.006. Epub 2013 Aug 8.


    Andrew J. Deans, Stephen C. West; DNA interstrand crosslink repair and cancer. Nat Rev Cancer. 2011 June 24; 11(7): 467–480. 


    Joseph San Filippo, Patrick Sung, and Hannah Klein;  Mechanism of Eukaryotic Homologous Recombination. Annu. Rev. Biochem. 2008. 77:229–57.


    Gene cards: http://www.genecards.org/cgi-bin/carddisp.pl?gene=FANCM


    Angelo Guainazzi, Orlando D. Schärer; Using synthetic DNA interstrand crosslinks to elucidate repair pathways and identify new therapeutic targets for cancer chemotherapy. Cell Mol Life Sci. 2010 November; 67(21): 3683–3697. 


    Pekka Katajisto, Julia Döhla, Christine L. Chaffer, Nalle Pentinmikko, Nemanja Marjanovic, Sharif Iqbal, Roberto Zoncu, Walter Chen, Robert A. Weinberg, and David M. Sabatini; Asymmetric apportioning of aged mitochondria between daughter cells is required for stemness. Science 17 April 2015: 340-343. 
    Published online 2 April 2015 [DOI:10.1126/science.1260384] - Stem cells preferentially select new rather than old mitochondria as they divide.


    Kim YJ, Wilson DM 3rd.; Overview of base excision repair biochemistry. Curr Mol Pharmacol. 2012 Jan;5(1):3-13.


    Ann-Christin Lindås & Rolf Bernander; The cell cycle of archaea. Nature Reviews Microbiology 11, 627-638 (2013). doi:10.1038/nrmicro3077.


    Alena V. Makarova, Peter M. Burgers; Eukaryotic DNA polymerase ζ.  DNA Repair, Volume 29, May 2015, Pages 47–55.


    Mukherjee S, Guainazzi A, Schärer OD; Synthesis of structurally diverse major groove DNA interstrand crosslinks using three different aldehyde precursors. Nucleic Acids Res. 2014 Jun;42(11):7429-35. doi: 10.1093/nar/gku328. Epub 2014 Apr 29.


    Parameswary Muniandy, Jia Liu, Alokes Majumdar, Su-ting Liu, and Michael M. Seidman; DNA INTERSTRAND CROSSLINK REPAIR IN MAMMALIAN CELLS: STEP BY STEP. Crit Rev Biochem Mol Biol. 2010 February ; 45(1): 23–49. doi:10.3109/10409230903501819.


    David M. Noll, Tracey McGregor Mason, and Paul S. Miller; Formation and Repair of Interstrand Cross-Links in DNA.  Chem Rev. 2006 February; 106(2): 277–301


    O'Flaherty DK, Denisov AY, Noronha AM, Wilds CJ.; NMR structure of an ethylene interstrand cross-linked DNA which mimics the lesion formed by 1,3-bis(2-chloroethyl)-1-
    nitrosourea. ChemMedChem. 2014 Sep;9(9):2099-103. doi: 10.1002/cmdc.201402121. Epub 2014 Jun 16. PMID: 24931822.


    Adisorn Ratanaphan;  A DNA Repair BRCA1 Estrogen Receptor and Targeted Therapy in Breast Cancer. Review. Int. J. Mol. Sci. 2012, 13(11), 14898-14916; doi:10.3390/ijms131114898


    Räschle M, Knipscheer P, Enoiu M, Angelov T, Sun J, Griffith JD, Ellenberger TE, Schärer OD, Walter JC; Mechanism of replication-coupled DNA interstrand crosslink repair. Cell. 2008 Sep 19;134(6):969-80. doi: 10.1016/j.cell.2008.08.030.


    Vogel EW, Barbin A, Nivard MJ, Stack HF, Waters MD, Lohman PH.; Heritable and cancer risks of exposures to anticancer drugs: inter-species comparisons of covalent deoxyribonucleic acid-binding agents. Mutat Res. 1998 May 25; 400(1-2):509-40.

     


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    Bioconjugation Chemistries for Molecular Engineering


    Recent developments in protein engineering, also known as bioengineering, have enabled the production of modified biomolecules such as proteins, DNA and RNA oligonucleotides containing desired properties useful for the creation of novel applications and nano-devices. During the last decades, chemical conjugation reactions have been applied in biology, molecular biology, and molecular medicine for various applications.

    Bioconjugation chemistry is a research field that studies the linking of one molecule to another by chemical or biological means. Typically, the resulting complexes are formed from at least one biomolecule, however, several molecules may be conjugated together as well. In addition, purely synthetic conjugated molecules are possible as well.  

    The study and use of chemical conjugation reactions, now also known as bioconjugation reactions, has more recently evolved into an important research topic as can be monitored by the number of publications in this field. For example, site-specific bioconjugations of a multitude of biomolecules to proteins, DNA, RNA, and carbohydrates, or to each other, have been developed. The resulting conjugates are useful for applications such as ligand discovery, disease diagnosis, and high-throughput screening, in vivo imaging, sensing, catalysis, therapeutics, as well as cell targeting. More recently, polymer brushes linked with biotin moieties allowing for the development of streptavidin-mediated conjugation capture agents in NanoVelcro chips have been engineered. These conjugates are a new type of molecular probes for prenatal diagnostics (GEN July 2015).  




    General bioconjugation chemistry schemes are illustrated in f
    igure 1. Typically, bioconjugation reactions are employed to couple biomolecules to surfaces or solid supports. Typical supports are beads, gold surfaces, nitrocellulose or dextran based arrays. The use of these chemistries allows the synthesis of oligonucleotide-, peptide-, or protein based libraries coupled to a solid support such as a micro-chip.  Typical applications are screening or sequencing by hybridization. The conjugation of oligo-nucleotides to a support is shown as an example. However, peptides, carbohydrates and proteins can also be used in a similar fashion. Other nanostructures such as dendrimers, cyclodextrin or cellulose, modified or unmodified, are also often utilized as spacers or amplifying moieties, usually between the support and the biomolecule selected for conjugation. 



    Figure 1: General schematic of bioconjugation chemistries. The conjugation of oligonucleotides to a solid support or surface is illustrated here. Three bioconjugation chemistries commonly used are reviewed. Carbonyl-diimidazole (CDI) activation to silanol groups is shown to the left. The surface functionalization technique using glycidooxypropyl-trimethoxy-silane (GOPS) is shown in the middle. And, the surface functionalization technique using 3-aminopropyltrimethoxysilane (APTMS) is shown at the right. Dendrimers or other branched polymers or other spacer molecules can also be used, often between the support and the molecule to be conjugated. 


    Bioengineering or Biological engineering is a new scientific field that applies engineering principles to biological systems. Broadly viewed, bioengineering can include elements of electrical, mechanical, computer science, materials, chemistry, biology and medical biology. Its main goal is to apply concepts and methods observed in biological systems to solve real-world problems in life sciences. Often medical biology is part of this endeavor. Bioengineering uses primarily knowledge gained from the fast developing field called “molecular biology.” A rapid development of many innovative techniques and methodologies pertinent to biological or medical applications using bioengineering principles leading to new applications in medicine, agriculture, and energy or electronic production occurred in recent decades. A new branch known as “biomimetics” strives to engineer new materials to mimic structures and functions of molecules found in living organisms using DNA, RNA, and protein molecules. The goal is the production or manufacture of new types of nano-materials, such as hydrogels,nano-particles, artificial proteins, antibodies, peptides, or dendrimers, and many others. However, the success of each conjugation reaction depends highly on its chemoselectivity under physiological conditions. Therefore, various chemoselective cross-linkers have been developed for the purposes of labeling and conjugation of selected molecules to functional groups available on target molecules. The following paragraphs describe several of these bioconjugation chemistries.

     

    1)    Formation of amide bonds, including urea and thiourea moieties.

    Typically amide bonds are formed through the reaction of an amino group with a carboxylic acid. Activator reagents are utilized to form the bonds more efficiently. Activator molecules such as 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), carbonyl-diimidazole (CDI) or similar compounds are commonly used as illustrated in figure 2A. Figures 2B and 2C show the reaction of the amino group with active esters. The reaction of amino groups with isocyanate or isothiocyanate produces a urea or thiourea backbone, respectively, as depicted in figure 1D. Many biomolecules including proteins have many amino and carboxyl groups at their molecular surface.  Also, amino and carboxyl groups can be introduced to synthetic oligonucleotides and peptides with ease. Therefore, these chemistries are most widely used for the production of bioconjugates. Even some newly developed cross-linkers, for example, copper-free click chemistry based cross-linkers, use these types of reaction known to organic chemists for a long time, to modify biomolecules. Under standard conditions, these chemical reactions are normally quick and selective, forming stable amide bonds within a few hours. However, the majority of conjugation reactions are normally performed in neutral or slightly basic conditions, free of primary and secondary amines. EDC-mediated conjugations are an exception. In this case, reactions are best performed at slightly acidic conditions. Amide forming reactions are normally selective towards amino groups and are thermodynamically or kinetically controlled. However, isocyanates and isothiocyanates also react with the hydroxyl group to form carbamate and thiocarbamate bonds, respectively. Thus, these cross-linkers are also used to conjugate hydroxyl-containing molecules.


    Figure 2. Amide formation reactions frequently used in bioconjugation.


    2)    Formation of thioethers

    The formation of thioesters is a widely used conjugation chemistry known for quite some time now. Reactions are very selective and specific since thiol groups are available in proteins that contain cysteine.  Some proteins, such as members of the C3/α-2M thioester protein family, and peptides that contain lanthionine, contain natural interchain thioester bonds. The formation of thiols bonds in biomolecules is relative straight forward. Also, incorporation of thiol groups into synthetic oligonucleotides and peptides is done with the help of standard chemistries. An advantage of the thioester chemistry is that primary amines do not interfere with the reaction avoiding the formation of byproducts. Also, reactions can be performed at a broad pH range, ranging from pH 2 to 10. Therefore, many hetero-bifunctional cross-linkers are typically designed to contain one thiol-reactive group andoneamino-reactive group. Reactive compounds that form thioether bonds or linkages include thiol-maleimide (Figure 3A), and thiol-haloacetate (Figure 3B). The lesser electronegativity between the sulfur and hydrogen atoms, compared to the oxygen and hydrogen atoms, make the thiol group less polar than the hydroxyl group. The reductive dealkylation of thioethers generates thiols.

     

     Figure 3. Frequently used bioconjugation thioether formation reactions.


    3)    Conjugation reactions involving carbonyl group

    The presence of carbonyl groups in biomolecules is very limited. However, these are important functional groups allowing the conjugation of saccharide moieties. Except for the aldehyde and ketone groups of the linear saccharides, hydroxyl groups at the anomeric position of the cyclic saccharides normally are not reactive enough and, therefore, are not available for bioconjugation reactions. In particular, 1,2-diol groups in saccharides can be specifically oxidized to aldehydes with the help of sodium periodate.  Carbonyl groups can be introduced to biomolecules using cross-linkers such as N-succinimidyl-4-formylbenzamide (S-4FB).  Carbonyl groups are reactive towards primary amines, including hydrazine and oxyamine groups, whereas thiol and hydroxyl groups usually don’t interfere with the reaction. The reaction of a carboxyl group with a primary amine, hydrazine or oxyamine form Schiff bases, hydrazone or oxime moieties, respectively, as illustrated in figure 4. Oxime groups are quite stable and exist as two stereoisomers. However, Schiff bases and hydrazones are more labile, especially in acidic solutions or conditions. Therefore, the Schiff base is typically reduced to a secondary amine with the help of sodium cyanoborohydride, called reductive amination, as depicted in figure 4A. Furthermore, hydrazones can also be reduced and are typically stabilized by adding an aromatic group or groups to the resulting compounds in proximity to the hydrazone group.

     

    Figure 4. Conjugation reactions involving carbonyl groups.

     

    1)          Thiol-exchange reactions

    Thiol-exchange reactions are widely used because these reactions produce cleavable conjugates.  Disulfide bonds are created between the conjugated molecules. Disulfide bonds containing conjugates can be cleaved via reduction by thiol reductases in tissue or cells. Furthermore, disulfide bonds can also be chemically cleaved in vitro using reducing agents such as mercaptoethanol (2-ME), tris(2-carboethyl)phosphine (TCEP), or dithiothreitol (DTT). However, the reactions for the formation of conjugates are usually slow but very selective towards the thiolated molecules. Amino or hydroxyl groups don’t usually interfere to produce byproducts. Although sometimes methanethiosulfonate reagents are used, 2-pyridyldithiol reagents are the most widely used thio-exchange cross-linkers, illustrated in figure 5. 



    Figure 5. The thiol-exchange reaction of 2-pyridyldithiol based cross-linkers.


    5)    Click chemistry and tetrazine ligation

    Click chemistry has gained a huge momentum in bioconjugation due the development of the reagents recently.  Traditionally, click chemistry refers to the reaction of an alkynyl compound with an azido compound by the catalysis of Cu(I) (Figure 6A). Although this reaction is very selective and clean, coordination of Cu(I) with many ligands makes the complete removal from the conjugate problematic. Furthermore, the toxicity of copper limits the application of click chemistry in many conjugation chemistries.  Copper-free click chemistry overcomes this problem. Here, cyclooctyne is used instead of common alkynes, the tension in the 8-member ring promotes the click reaction,  no longer requiring Cu(I) catalysis.  Also, since recently needed reagents became available at a lower price, the copper-free click chemistry became very popular in recent years. Frequently-used cyclooctynyl reagents include the cross-linkers dibenzocyclooctynyl (DBCO) and bicyclo[6.1.0]nonynyl (BCN).  In addition, the Diels-Alder reaction and reverse Diels-Alder reaction has been studied for years as a conjugation chemistry. A recent successful development is the conjugation reaction of trans-cyclooctene to tetrazines, called, a tetrazine ligation (Figure 6B). Here, the tension in the trans-cyclooctene ring allows the reaction to occur at room temperature, relatively fast and without the need for a catalyst. 


    Figure 6. Click  chemistry (A) and tetrazine ligation (B)


    6)    Photoreactive cross-linkers

    Photoreactions are only frequently performed in bioconjugation reactions. However, they are occasionally used for in-vivo or in-vitro crosslinking reactions.  Photoreactive cross-linkers commonly used are azidophenyl or diazirine compounds. Upon exposure to ultraviolet light, these compounds are activated and react with electron-donating groups, most frequently amino group, to produce conjugates (Figure 5). The reactions are normally not very fast and unselective, and may have multiple pathways. Also, products are not very clean. One advantage of photocrosslinking is that biomolecules that are selected to be conjugated can be mixed prior to the conjugation reaction, and the start of the reaction can be controlled.

    Figure 7.Photoreactions frequently used in bioconjugation.


    References

    Advances in Bioconjugation:   http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2901115/

    BC Bioconjugate Chemistry Journal:   
    http://pubs.acs.org/journal/bcches

    Chemical modifications and bioconjugate reactions of nanomaterials for sensing, imaging, drug delivery and therapy                                    






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    Small saccharides are not normally immunogenic, unless they are conjugated to a carrier protein. Hence, they are usually linked to a protein in ways that the structural integrity of the saccharide is maintained.

    Structural Integrity

    Carbohydrates are normally found in aqueous solutions as cyclic structures, i.e. pyranoside or furanoside types of rings. However, under certain conditions, these rings can open to yield linear chains; forms that are not found naturally.  Also, chemical modifications such as oxidation can open the rings to deliver new derivatives and allow high degree of conjugation. These changes would impact recognition of the sugars by antibodies and cellular receptors, i.e. lectins, making them physiologically and/or immunologically ineffective.

    What protein carriers can be used?

    In principle any protein that is non-glycosylated is fine as a carrier. Proteins that are glycosylated as KLH and ovalbumin are not recommended, as their oligosaccharides may also induce the formation of antibodies, which would interfere with the screening process, in the case of looking for a specific monoclonal. Proteins as tetanus toxoids, edestin and serum albumin are recommended as carriers.

    What density of the ligand is desirable?

    While normally we would like to have 20 to 30 sugar residues per mole of protein, the binding is usually in the order of 4 to 10 residues per mole of protein which is sufficient to induce an effective immune response.

    How Bio-Synthesis perform the conjugation?

    In principle we try to avoid procedures that alter the cyclic nature of the sugars. While somewhat more complicated, these methods deliver conjugates where the sugars are present as they are found normally in the body and not as a derivative that maybe physiologically non-functional.

    Types of chemistry used by Bio-Synthesis

    The selected chemical approach depends on the sugar in question. We look for groups that allow chemical modifications without altering the overall sugar structure, while avoiding procedures that will destroy their native characteristics.


    0 0

    Bioconjugation Chemistry for Molecular Engineering


    Recent developments in protein engineering, also known as bioengineering, have enabled the production of modified biomolecules such as proteins, DNA and RNA oligonucleotides containing desired properties useful for the creation of novel applications and nano-devices. During the last decades, chemical conjugation reactions have been applied in biology, molecular biology, and molecular medicine for various applications.

    Bioconjugation chemistry is a research field that studies the linking of one molecule to another by chemical or biological means. Typically, the resulting complexes are formed from at least one biomolecule, however, several molecules may be conjugated together as well. In addition, purely synthetic conjugated molecules are possible as well.  

    The study and use of chemical conjugation reactions, now also known as bioconjugation reactions, has more recently evolved into an important research topic as can be monitored by the number of publications in this field. For example, site-specific bioconjugations of a multitude of biomolecules to proteins, DNA, RNA, and carbohydrates, or to each other, have been developed. The resulting conjugates are useful for applications such as ligand discovery, disease diagnosis, and high-throughput screening, in vivo imaging, sensing, catalysis, therapeutics, as well as cell targeting. More recently, polymer brushes linked with biotin moieties allowing for the development of streptavidin-mediated conjugation capture agents in NanoVelcro chips have been engineered. These conjugates are a new type of molecular probes for prenatal diagnostics (GEN July 2015).  




    General bioconjugation chemistry schemes are illustrated in f
    igure 1. Typically, bioconjugation reactions are employed to couple biomolecules to surfaces or solid supports. Typical supports are beads, gold surfaces, nitrocellulose or dextran based arrays. The use of these chemistries allows the synthesis of oligonucleotide-, peptide-, or protein based libraries coupled to a solid support such as a micro-chip.  Typical applications are screening or sequencing by hybridization. The conjugation of oligo-nucleotides to a support is shown as an example. However, peptides, carbohydrates and proteins can also be used in a similar fashion. Other nanostructures such as dendrimers, cyclodextrin or cellulose, modified or unmodified, are also often utilized as spacers or amplifying moieties, usually between the support and the biomolecule selected for conjugation. 



    Figure 1: General schematic of bioconjugation chemistries. The conjugation of oligonucleotides to a solid support or surface is illustrated here. Three bioconjugation chemistries commonly used are reviewed. Carbonyl-diimidazole (CDI) activation to silanol groups is shown to the left. The surface functionalization technique using glycidooxypropyl-trimethoxy-silane (GOPS) is shown in the middle. And, the surface functionalization technique using 3-aminopropyltrimethoxysilane (APTMS) is shown at the right. Dendrimers or other branched polymers or other spacer molecules can also be used, often between the support and the molecule to be conjugated. 


    Bioengineering or Biological engineering is a new scientific field that applies engineering principles to biological systems. Broadly viewed, bioengineering can include elements of electrical, mechanical, computer science, materials, chemistry, biology and medical biology. Its main goal is to apply concepts and methods observed in biological systems to solve real-world problems in life sciences. Often medical biology is part of this endeavor. Bioengineering uses primarily knowledge gained from the fast developing field called “molecular biology.” A rapid development of many innovative techniques and methodologies pertinent to biological or medical applications using bioengineering principles leading to new applications in medicine, agriculture, and energy or electronic production occurred in recent decades. A new branch known as “biomimetics” strives to engineer new materials to mimic structures and functions of molecules found in living organisms using DNA, RNA, and protein molecules. The goal is the production or manufacture of new types of nano-materials, such as hydrogels,nano-particles, artificial proteins, antibodies, peptides, or dendrimers, and many others. However, the success of each conjugation reaction depends highly on its chemoselectivity under physiological conditions. Therefore, various chemoselective cross-linkers have been developed for the purposes of labeling and conjugation of selected molecules to functional groups available on target molecules. The following paragraphs describe several of these bioconjugation chemistries.

     

    1)    Formation of amide bonds, including urea and thiourea moieties.

    Typically amide bonds are formed through the reaction of an amino group with a carboxylic acid. Activator reagents are utilized to form the bonds more efficiently. Activator molecules such as 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), carbonyl-diimidazole (CDI) or similar compounds are commonly used as illustrated in figure 2A. Figures 2B and 2C show the reaction of the amino group with active esters. The reaction of amino groups with isocyanate or isothiocyanate produces a urea or thiourea backbone, respectively, as depicted in figure 1D. Many biomolecules including proteins have many amino and carboxyl groups at their molecular surface.  Also, amino and carboxyl groups can be introduced to synthetic oligonucleotides and peptides with ease. Therefore, these chemistries are most widely used for the production of bioconjugates. Even some newly developed cross-linkers, for example, copper-free click chemistry based cross-linkers, use these types of reaction known to organic chemists for a long time, to modify biomolecules. Under standard conditions, these chemical reactions are normally quick and selective, forming stable amide bonds within a few hours. However, the majority of conjugation reactions are normally performed in neutral or slightly basic conditions, free of primary and secondary amines. EDC-mediated conjugations are an exception. In this case, reactions are best performed at slightly acidic conditions. Amide forming reactions are normally selective towards amino groups and are thermodynamically or kinetically controlled. However, isocyanates and isothiocyanates also react with the hydroxyl group to form carbamate and thiocarbamate bonds, respectively. Thus, these cross-linkers are also used to conjugate hydroxyl-containing molecules.


    Figure 2. Amide formation reactions frequently used in bioconjugation.


    2)    Formation of thioethers

    The formation of thioesters is a widely used conjugation chemistry known for quite some time now. Reactions are very selective and specific since thiol groups are available in proteins that contain cysteine.  Some proteins, such as members of the C3/α-2M thioester protein family, and peptides that contain lanthionine, contain natural interchain thioester bonds. The formation of thiols bonds in biomolecules is relative straight forward. Also, incorporation of thiol groups into synthetic oligonucleotides and peptides is done with the help of standard chemistries. An advantage of the thioester chemistry is that primary amines do not interfere with the reaction avoiding the formation of byproducts. Also, reactions can be performed at a broad pH range, ranging from pH 2 to 10. Therefore, many hetero-bifunctional cross-linkers are typically designed to contain one thiol-reactive group andoneamino-reactive group. Reactive compounds that form thioether bonds or linkages include thiol-maleimide (Figure 3A), and thiol-haloacetate (Figure 3B). The lesser electronegativity between the sulfur and hydrogen atoms, compared to the oxygen and hydrogen atoms, make the thiol group less polar than the hydroxyl group. The reductive dealkylation of thioethers generates thiols.

     

     Figure 3. Frequently used bioconjugation thioether formation reactions.


    3)    Conjugation reactions involving carbonyl group

    The presence of carbonyl groups in biomolecules is very limited. However, these are important functional groups allowing the conjugation of saccharide moieties. Except for the aldehyde and ketone groups of the linear saccharides, hydroxyl groups at the anomeric position of the cyclic saccharides normally are not reactive enough and, therefore, are not available for bioconjugation reactions. In particular, 1,2-diol groups in saccharides can be specifically oxidized to aldehydes with the help of sodium periodate.  Carbonyl groups can be introduced to biomolecules using cross-linkers such as N-succinimidyl-4-formylbenzamide (S-4FB).  Carbonyl groups are reactive towards primary amines, including hydrazine and oxyamine groups, whereas thiol and hydroxyl groups usually don’t interfere with the reaction. The reaction of a carboxyl group with a primary amine, hydrazine or oxyamine form Schiff bases, hydrazone or oxime moieties, respectively, as illustrated in figure 4. Oxime groups are quite stable and exist as two stereoisomers. However, Schiff bases and hydrazones are more labile, especially in acidic solutions or conditions. Therefore, the Schiff base is typically reduced to a secondary amine with the help of sodium cyanoborohydride, called reductive amination, as depicted in figure 4A. Furthermore, hydrazones can also be reduced and are typically stabilized by adding an aromatic group or groups to the resulting compounds in proximity to the hydrazone group.

     

    Figure 4. Conjugation reactions involving carbonyl groups.

     

    1)          Thiol-exchange reactions

    Thiol-exchange reactions are widely used because these reactions produce cleavable conjugates.  Disulfide bonds are created between the conjugated molecules. Disulfide bonds containing conjugates can be cleaved via reduction by thiol reductases in tissue or cells. Furthermore, disulfide bonds can also be chemically cleaved in vitro using reducing agents such as mercaptoethanol (2-ME), tris(2-carboethyl)phosphine (TCEP), or dithiothreitol (DTT). However, the reactions for the formation of conjugates are usually slow but very selective towards the thiolated molecules. Amino or hydroxyl groups don’t usually interfere to produce byproducts. Although sometimes methanethiosulfonate reagents are used, 2-pyridyldithiol reagents are the most widely used thio-exchange cross-linkers, illustrated in figure 5. 



    Figure 5. The thiol-exchange reaction of 2-pyridyldithiol based cross-linkers.


    5)    Click chemistry and tetrazine ligation

    Click chemistry has gained a huge momentum in bioconjugation due the development of the reagents recently.  Traditionally, click chemistry refers to the reaction of an alkynyl compound with an azido compound by the catalysis of Cu(I) (Figure 6A). Although this reaction is very selective and clean, coordination of Cu(I) with many ligands makes the complete removal from the conjugate problematic. Furthermore, the toxicity of copper limits the application of click chemistry in many conjugation chemistries.  Copper-free click chemistry overcomes this problem. Here, cyclooctyne is used instead of common alkynes, the tension in the 8-member ring promotes the click reaction,  no longer requiring Cu(I) catalysis.  Also, since recently needed reagents became available at a lower price, the copper-free click chemistry became very popular in recent years. Frequently-used cyclooctynyl reagents include the cross-linkers dibenzocyclooctynyl (DBCO) and bicyclo[6.1.0]nonynyl (BCN).  In addition, the Diels-Alder reaction and reverse Diels-Alder reaction has been studied for years as a conjugation chemistry. A recent successful development is the conjugation reaction of trans-cyclooctene to tetrazines, called, a tetrazine ligation (Figure 6B). Here, the tension in the trans-cyclooctene ring allows the reaction to occur at room temperature, relatively fast and without the need for a catalyst. 


    Figure 6. Click  chemistry (A) and tetrazine ligation (B)


    6)    Photoreactive cross-linkers

    Photoreactions are only frequently performed in bioconjugation reactions. However, they are occasionally used for in-vivo or in-vitro crosslinking reactions.  Photoreactive cross-linkers commonly used are azidophenyl or diazirine compounds. Upon exposure to ultraviolet light, these compounds are activated and react with electron-donating groups, most frequently amino group, to produce conjugates (Figure 5). The reactions are normally not very fast and unselective, and may have multiple pathways. Also, products are not very clean. One advantage of photocrosslinking is that biomolecules that are selected to be conjugated can be mixed prior to the conjugation reaction, and the start of the reaction can be controlled.

    Figure 7.Photoreactions frequently used in bioconjugation.


    References

    Advances in Bioconjugation:   http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2901115/

    BC Bioconjugate Chemistry Journal:   
    http://pubs.acs.org/journal/bcches

    Chemical modifications and bioconjugate reactions of nanomaterials for sensing, imaging, drug delivery and therapy                                    






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    A new BNA-NC-DNA-peptide conjugate as a potential treatment for multi-drug resistant infections.


    A new type of BNA/DNA-peptide conjugate has been developed for the treatment of drug-resistant infections. This new 
    BNA/DNA-peptide conjugate is thought to become a useful antimicrobial and chemotherapeutic agent.



    Lopez et al. (2015) recently 
    published a paper where a new type of BNANC-DNA-peptide conjugate was investigated for its use as an antisense agent for the treatment of amikacin resistant infections. The research group tested a BNANC-DNA hybrid co-oligomer conjugated to a cell permeable peptide mimic named CPPBD4 for its ability to reduce the level of resistance in A. baumannii A 155 cells.


    “Christina Lopez, Brock A. Arivett, Luis A. Actis, Marcelo E. Tolmasky;

    Inhibition of AAC(6')-Ib-Mediated Resistance to Amikacin in Acinetobacter baumannii by an Antisense Peptide-Conjugated 2’,4’-Bridged Nucleic Acid-NC-DNA Hybrid Oligomer. Antimicrobial Agents and Chemotherapy. September 2015 Volume 59 Number 9. http://aac.asm.org/content/early/2015/07/07/AAC.01304-15.abstract


    The conjugation of compounds such as oligonucleotides or oligonucleotide mimetics to cell-penetrating peptides is a now a well known strategy to guide antisense oligomers inside cells. These peptides contain a small number of amino acids, usually less than 30, are amphipathic, and have a net positive charge. Using this strategy, Lopez at al. used BNAs for the design of an antisense inhibitor peptide-conjugate to decrease the resistance to amikacin as mediated by AAC(6’)-Ib, a wide-spread aminoglycoside-modifying enzyme.

    The use of CPPBD4 in combination with amikacin for the treatment of infected larvae of the greater wax moth Galleria mellonella lowered the level of resistance to amikacin. The result was that the larvae showed a significant increase in survival rates. The reported observed survival rates were similar to the survival rates of non-infected larvae. Reported results indicated that the hybrid analogs composed of BNANC and DNA conjugated to a cell-permeable peptide reached the cytosol of A. baumannii where it exhibited an antisense effect. The peptide-BNANC-conjugate investigated is reported to be useful for the treatment of amikacin resistant bacterial infections.

    The antisense peptide-BNA-oligonucleotide conjugate targets the aminoglycoside 6’-N-acetyltransferase type Ib [AAC(6’)-Ib] gene from A. baumannii by binding to nucleotide sequences around the initiation codon of this gene. The chemical structure and a model of a BNANC(NMe) residue is shown in figure 1.


    Figure 1: Chemical structure and model of a 2’,4’-BNANC residue.

    2,4-Bridged nucleic acid-NC (BNANC) analogs exhibit beneficial features such as higher binding affinity to a cRNA, excellent single-mismatch discriminating ability, and tests carried out in mice showed that BNANC-based antisense molecules have minimal toxicity. The 2’,4’-BNANC antisense oligonucleotide peptide-conjugate CPPBD4 inhibits the growth of A. Baumani A155 thereby inhibiting bacterial infection. Lopez et al investigated the 2’,4’-BNANC antisense oligonucleotide conjugate CPPBD4 for its use of inhibiting the expression of resistance to amikacin (AMK) in A. Baumani A155.

    CPPBD4


    The peptide BNA-conjugate used for the study

    (RXR)4XB-Cys-SMCC-C6 amino-2’,4’-BNANC-DNA

    (RXR)4XB-Cys-SMCC-C6 amino-C+TGCT+GCGT+AACA+TC

    + indicates the location of the BNA monomers; R = arginine; X = 6-aminohexanoic acid; B = β-alanine; SMCC = succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate.

    Targets the nucleotide sequence depicted in figure 2.


    Figure 2: Nucleotide sequences targeted by the antisense BNANC-DNA peptide conjugate. The nucleotide sequences around the initiation codon of the A. baumanii (Ab) A155 aac(6’)-IB gene are shown at the bottom (Lopez at al. 2015). The nucleotide sequence with the location of the BNA monomers (in magenta) are shown at the top.


    Acinetobacter baumannii 

    Acinetobacter baumannii is a short, rod-shaped Gram-negative bacterium that can be an opportunistic pathogen in humans. People with compromised immune systems are often affected by it. A. baumannii is increasingly becoming important in hospital-derived infections. The bacteria can also be found in the environment, for example in various soils and in water.

    The A. baumannii A155 strain was isolated from a urinary sample. This strain harbors the aac(6)-Ib gene which is the most common amikacin (AMK) resistance

    gene found in Gram-negative pathogens.

    Hospital-acquired infections (HAI) or nosocomial infections are infection acquired during the stay in a hospital or an infection developed by hospital staff. The World Health Organization defines them as follows

    “Infections acquired in a hospital by a patient who was admitted for a reason other than infection. An infection occurring in a patient in a hospital, or other health care facility, in whom the infection was not present, or incubating at the time of admission. This includes infections acquired in the hospital but appearing after discharge, and also occupational infections among staff of the facility.”

    Amikacin

    Amikacin is an aminoglycoside antibiotic used for the treatment of different bacterial infections. Amikacin is often used for the treatment of severe, hospital-acquired infections caused by multidrug-resistant Gram-negative bacteria. Usually, amikacin is used as a last-resort medication against multidrug-resistant bacteria.
    (Source:
    http://www.toku-e.com/Assets/MIC/Amikacin%20hydrate.pdf).

    Amikacin is an aminoglycoside antibiotic derived from kanamycin A that similar to other aminoglycosides disrupts bacterial protein synthesis by binding to the 30S ribosome of susceptible organisms. Binding interferes with mRNA binding and tRNA acceptor sites leading to the production of non-functional or toxic peptides. Other mechanisms not fully understood may confer the bactericidal effects of amikacin. Amikacin is also nephrotoxic and ototoxic. 


    Figure 3: Structures and Models of Amikacin (Source:  Drugbank http://www.drugbank.ca/drugs/DB00479).


    Aminoglycosides

    Aminoglycosides are very effective tools to fight infections but unfortunately resistance levels are growing. Over the years bacteria have developed numerous mechanisms to resist the actions of aminoglycosides. Bacteria can use enzymatic modifications to defeat the actions of antibiotics. The aminoglycoside 6’-N-acetyltransferase type Ib [AAC(6’)-Ib] is of great clinical relevance and is found in over 70% of AAC(6)-I-producing gram-negative clinical isolates therefore it has been the subject of numerous studies. The resistance is due to synthesis of the aminoglycoside 6'-N-acetyltransferase type I [AAC(6')-I] enzyme which modifies amikacin and tobramycin. AAC(6′)-Ib (aminoglycoside 6′-N-acetyltransferase type Ib) is found in a wide variety of gram-negative pathogens. The enzyme has significant microheterogeneity at its N-termini and the aac(6′)-Ib gene is often present in integrons, transposons, plasmids, genomic islands, and other genetic structures.


    Since the study of bacterial infections requires animal models and mammalian models of infections are costly, insect infection models have been shown to provide valuable alternatives. Larvae of the greater wax moth Galleria mellonella have been shown to provide valuable insight into pathogenesis of many microbial infections.  Results obtained with Galleria larvae infected by direct injection through the cuticle are reported to consistently correlate with those of similar mammalian studies and are therefore now used as a powerful infection model system.



    Reference

    Weblinks

    http://www.uphs.upenn.edu/bugdrug/antibiotic_manual/aminoglycosideresistance.htm

    http://emedicine.medscape.com/article/236891-overview

    http://www.cdc.gov/HAI/organisms/acinetobacter.html


    Christina Lopez,
    Brock A. Arivett,Luis A. Actis,Marcelo E. TolmaskyInhibition of AAC(6=)-Ib-Mediated Resistance to Amikacin in Acinetobacter baumannii by an Antisense Peptide-Conjugated 2,4-

    Bridged Nucleic Acid-NC-DNA Hybrid Oligomer. Antimicrobial Agents and Chemotherapy. September 2015 Volume 59 Number 9.


    Rahman SM, Seki S, Utsuki K, Obika S, Miyashita K, Imanishi T.; 
    2007. 2',4'-BNA(NC): a novel bridged nucleic acid analogue with excellent hybridizing and nuclease resistance profiles. Nucleosides Nucleotides. Nucleic Acids 26:1625–1628. http://dx.doi.org/10.1080/15257770701548980.

    Ramarao N, Nielsen-Leroux C, Lereclus D.; The insect Galleria mellonella as a powerful infection model to investigate bacterial pathogenesis. J Vis Exp. 2012 Dec 11;(70):e4392. doi: 10.3791/4392.


    María S. Ramirez, Nikolas Nikolaidis and Marcelo E. Tolmasky
    ;  Rise and dissemination of aminoglycoside resistance: the aac(6′)-Ib paradigm. Front. Microbiol., 17 May 2013 | http://dx.doi.org/10.3389/fmicb.2013.00121http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3656343/pdf/fmicb-04-00121.pdf

    Matthew W. Vetting, Luiz Pedro S. de Carvalho, Michael Yu, Subray S. Hegde, Sophie Magnet, Steven L. Roderick, John S. Blanchard; Minireview: Structure and functions of the GNAT superfamilyof acetyltransferases. Archives of Biochemistry and Biophysics 433 (2005) 212–226.


    Yamamoto T, Harada-Shiba M, Nakatani M, Wada S, Yasuhara H, 
    Narukawa K, Sasaki K, Shibata MA, Torigoe H, Yamaoka T, Imanishi, T, Obika S. 2012. Cholesterol-lowering action of BNA-based antisense oligonucleotides targeting PCSK9 in atherogenic diet-induced hypercholesterolemic mice. Mol Ther Nucleic Acids 1:e22. http://dx.doi.org/10.1038/mtna.2012.16.


    WHO/CDS/CSR/EPH/2002.12: Prevention of hospital-aquired infections. A practical guide 2nd edition.  (Source:
    http://www.who.int/emc).


    Appendix


    GCN5-related N-acetyltransferase

    Over more than 150 distinct enzymes found in the kingdom of life appear to have evolved from a common ancestral N-acetyltransferase (NAT). Wolf et al. in 1998 reported the structure for the canonical GCN5-related N-acetytransferase. This enzyme belongs to the GCN5-related NATs or GNAT super family. The GCN5-related N-acetytransferases are enzymes that are universally distributed in nature. The enzymes use acyl-CoAs for the acylation of their substrates. The structure of the GCN5-related N-acetyltransferase is shown in the figure below. One subgroup of these enzymes, a eubacterial aminobglycoside N-acetyltransferase, catalyzes the acetyl group addition to aminoglycoside antibiotics directed against aerobic, gram-negative bacilli. Serratia marcescens are rod-shaped gram-negative bacteria, a Enterobacteriaceae, that is a human pathogen involved in hospital-acquired infections. This enzyme has the same N-terminal end as the A. baumannii A 155 aac(6’)-1b gene product.



    Aminoglycoside 
    N-acetyltransferases  catalyze the regioselective acetylation of one of the four amino groups found on a diverse set of aminoglycosides having antibiotic properties. Acetylation of an amino group reduces the affinity of these compounds for the acceptor tRNA site on the 30S ribosome by four orders of magnitude. This makes bacteria expressing these genes resistant to antibiotics.

    Figure 4: Model of the GCN5-related N-acetyltransferase.In 1998 Wolf et al. determined the X-ray structure of a canonical GCN5-related N-acetyltransferase (GNAT), the Serratia marcescens aminoglycoside 3-N-acetyltransferase, bound to coenzyme A (CoA) at a 2.3 Å resolution. This enzyme belongs to the GNAT superfamily of proteins. 


    Reference

    Eva Wolf, Alex Vassilev, Yasutaka Makino, Andrej Sali, Yoshihiro Nakatani, Stephen K. BurleyCrystal Structure of a GCN5-Related N-acetyltransferase. Serratia marcescens Aminoglycoside 3-N-acetyltransferase. Volume 94, Issue 4, p439–449, 21 August 1998. DOI: http://dx.doi.org/10.1016/S0092-8674(00)81585-8.

    -.-


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    Tyrosine sulfation is a post-translational modification of proteins and peptides. Many secreted and trans-membrane proteins contain sulfated tyrosine residues, including chemokine receptors. The enzymes tyrosylprotein sulfotransferases or TPSTs (EC 2.8.2.20) catalyze the transfer of a sulfonate group from the donor compound 3’-phosphoadenosine 5’-phosphosulfate or PAPS to the hydroxyl group of a luminally oriented peptidyltyrosine residue to form a tyrosine O4-sulfate ester and 3’,5’-ADP. The reaction is illustrated in figure 1 below.

    Figure 1: The tyrosylprotein sulfotransferase reaction. The enzymes tyrosylprotein sulfotransferases or TPSTs (EC 2.8.2.20) catalyze the transfer of a sulfonate group from the donor compound 3’-phosphoadenosine 5’-phosphosulfate or PAPS to the hydroxyl group of a luminally oriented peptidyltyrosine residue to form a tyrosine O4-sulfate ester and 3’,5’-ADP.  

    The addition of a sulfate group (SO3) to a tyrosine peptide results in a mass shift of ~80 Da to a higher mass. This mass shift is nearly isobaric with that of a phosphate group due to phosphorylation (monoisotopic masses: SO3 79.9568; HPO3 79.9663). The very small difference of this mass difference results in an analytical challenge for the detection of this group when conjugated to tyrosine residues in proteins or peptides when using most mass spectrometers.

    However, the use of alkaline phosphatase allows the removal of phosphorylated peptides, leaving only sulfonated peptides for the analysis. Also, anti-sulfotyrosine antibodies allow for the selective enrichment of sulfotyrosine peptides thereby enabling the identification of these type of peptides.


    Synthetic sulfated tyrosine peptides containing sulfotyrosine can be utilized as substrates or model peptides for the study of sulfation kinetics and protein-peptide or protein-protein interactions. Optimized peptide synthesis strategies can be used to generate sulfated peptides. Simpson et al. in 2009 reported a peptide synthesis strategy for the synthesis of sulfotyrosine derivatives. The research group utilized the neopentyl protecting group for sulfate monoesters to achieve a high-yield synthesis of tyrosine-sulfated peptides.

    Receptor tyrosine sulfation enhances chemokine affinity in a site-specific manner thereby influencing chemokine interactions. A review article published by Ludeman and Stone in 2013 describes how sulfation of the same receptor peptide at different positions can have a different effect on chemokine binding affinity. Variations in peptide or protein sulfation may alter affinities of chemokines for their receptors resulting in a regulation of cellular responses to chemokines. These findings support the possibility that differential receptor sulfation can modulate chemokine selectivity and chemokine oligomerization.


    Table 1 contains a list of substrates and sulfated peptides, and table 2 contains info gained from biophysical studies of chemokine binding by receptor
    sulfopeptides.

    Table 1: Tyrosine substrate peptides or tyrosine sulfated peptides.

    (Source: Ludeman and Stone; 2014).

     

    Peptide

    Substrate

    MW

     

    HPO3 

    79.9663

     

    SO3

    79.9568

    C4P5y3

    EDFEDYEFD

    1,208.18

    C4P5y3

    EDFEDY(SO3H)EFD

    1,289.14

    Human complement C4

    EDYEDYEYD

    1,223.41

    Human complement C4

    EDYEDY(SO3H)EYD

    1,304.38

    Selectin P ligand, CRA b

    YEYLDYDF

    1,127.18

     

    YEY(SO3H)LDYDF

    1,208.14

     

    LDYDF

    671.71

     

    LDY(SO3H)DF

    752.66

     

    LDY(PO3)DF

    752.68

    Selectin P ligand, isoform CRA b

    QATEXEXLDXDFLPETEPP

     

    Psgl-1 peptide

    QATEXEXLDXDFLPETEPPRPMMDDDDK

     

     

    QATEYEYLDYDFLPETEPP

    2,320.53

     

    QATEY(SO3H)EYLDYDFLPETEPP

    2,401.5

     

    QATEYEY(SO3H)LDYDFLPETEPP

    2,401.5

     

    QATEYEYLDY(SO3H)DFLPETEPP

    2,401.5

     

    QATEY(SO3H)EY(SO3H)LDYDFLPETEPP

    2,382.46

     

    QATEY(SO3H)EY(SO3H)LDY(SO3H)DFLPETEPP

    2,482.44

     

    QATEYEYLDYDFLPETEPPRPMMDDDDK

    3,424.78

     

    QATEY(SO3H)EYLDYDFLPETEPPRPMMDDDDK

    3,505.74

     

    QATEYEY(SO3H)LDYDFLPETEPPRPMMDDDDK

    3,505.74

     

    QATEYEYLDY(SO3H)DFLPETEPPRPMMDDDDK

    3,505.74

     

    QATEY(SO3H)EY(SO3H)LDYDFLPETEPPRPMMDDDDK

    3,586.69

     

    QATEY(SO3H)EY(SO3H)LDY(SO3H)DFLPETEPPRPMMDDDDK

    3,667.65

     

     

    3,586.69

    Fibrinogen beta peptide, mouse

    ENENVINEYSSILEDQR

    2,052.23

     

    ENENVINEY(SO3H)SSILEDQR

    2,133.19

     

     

     

    Cholecytokinin (26-33)

    DYMGWMDF-NH2

    1,063.25

    Cholecytokinin (26-33)

    DY(SO3H)MGWMDF-NH2

    1,141.3

    Leu-Enkephalin

    YGGFL

    554.68

    Leu-Enkephalin

    Y(SO3H)GGFL

    634.2

    Hirudin

    DFEEIPEEYLQ

    1,409.7

    Hirudin

    DFEEIPEEY(SO3H)LQ

    1,489.6

     

     

     

    Model peptide

    KESDYLKNT

    1,096.25

    Model peptide

    KESDY(SO3H)LKNT

    1,177.2



    Table 2: Chemokine receptors known to be sulfated and their cognate chemokines.

    (Source: Ludeman and Stone; 2014).

    Receptor

    Chemokine ligands

    Receptor N-terminal amino acid sequence

    Key findings

    CCR2

    CCL2/MCP-1

    CCL7/MCP-3

    CCL8/MCP-2

    CCL11/eotaxin-1

    CCL13/MCP-4

    CCL16/HCC-4/LEC

    1MLSTSRSRFIRNTNESGEEVTTFFDYDYGAPC32

    Y26 is sulfated

    Y26A mutant has reduced receptor binding/activation.

    Mutation of D25 reduces sulfation.

    (Preobrazhensky et al.,

    2000; Tan et al., 2013)

    CCR5

    CCL3/MIP-1α

    CCL4/MIP-1β CCL5/RANTES

    CCL8/MCP-2

    CCL11/eotaxin-1

    CCL14/HCC-1

    CCL16/HCC-4/LEC

    1MDYQVSSPIYDINYYTSEPC20

    CCR5 is Tyr-sulfated.

    Sulfated Tyr residues contribute to binding

    of CCL3, CCL4 and HIV-1 surface

    proteins.

    (Farzan et al., 1999)

     

    CCR8

    CCL1/I-309

    CCL4/MIP-1β

    CCL16/ HCC-4/LEC

    CCL17/TARC

    1MDYTLDLSVTTVTDYYYPDIFSSPC25

    N-terminal Tyr residues are sulfated.

    Sulfated Tyr residues contribute to binding

    of I-309.

    (Gutierrez et al., 2004)

    CXCR3

    CXCL9/Mig

    CXCL10/IP-10

    CXCL11/I-TAC

     

    1MVLEVSDHQVLNDAEVAALLENFSSSYDYGENESDSC37

    Y27 and Y29 or CXCR3 are sulfated.

    Mutation of Y27 or Y29 reduces binding and activation by CXCL9-11.

    (Colvin et al., 2006; Gao

    et al., 2009)

    CXCR4

    CXCL12/SDF-1

    1MEGISIYTSDNYTEEMGSGDYDSMKEPC28

    N-terminal Tyr residues are sulfated.

    Mutation of N-terminal Tyr residues reduces CXCL12 binding.

    (Farzan et al., 2002a)

    CX3CR1

    CX3CL1/fractalkine

    1MDQFPESVTENFEYDDLAEACYIGDIV27

    Mutation of N-terminal Tyr residues or sulfatase treatment reduces fractalkine

    binding affinity.

    (Fong et al., 2002)

    DARC

    Many CC and CXC chemokines

    1MGNCLHRAELSPSTENSSQLDFEDVWNSSYGVNDSFP

    DGDYDANLEAAAPCHSCNLLDDS60

    Y30 and Y41 are sulfated.

    Mutation of Y30 and Y41 reduces binding

    to different chemokines.

    Mutation of Y41 reduces binding to

    Plasmodium vivaxDuffy binding protein.

    (Choe et al., 2005)

    Receptor Chemokine ligands1 Receptor N-terminal amino acid sequence2 Key findings References

    1Chemokine ligands are those listed in Szpakowska et al. (2012). 2Potentially sulfated Tyr residues are shown in bold; acidic residues are underlined.

     

    Table 2: Biophysical studies of chemokine binding by receptor sulfopeptides.

    (Ludeman and Stone; 2014).

    Receptor

    Chemokine(s) studied

    Sulfopeptide

    Key findings

    CCR2

    CCL2/MCP-1

    (wild type; obligate

    monomer P8A; obligate

    dimer T10C)

    18EEVTTFFDYDYGAP31

    (all four sulfation states)

     

    All three forms of CCL2 bind CCR2

    sulfopeptides.

    Sulfation of single Tyr residues enhances affinity by 4- to 30-fold.

    Sulfation of both Tyr residues enhances affinity additively for monomer and cooperatively for dimer.

    Sulfopeptides destabilize dimeric CCL2 in favour of active monomer.

    Sulfopeptides bind to N-loop/β3 site.

    (Tan et al., 2013)

    CCR2

    CCL7/MCP-3

    21TTFFDYDYGA30

    (non-sulfated, monosulfated and disulfated)

    Single sulfation enhances CCL7 affinity 4-fold.

    Double sulfation enhances CCL7 affinity 36-fold.

    Binding affinities can be measured by

    electrospray mass spectrometry.

    (Jen and Leary, 2010)

    CCR3

    CCL11/eotaxin-1

    CCL24/eotaxin-2

    CCL26/eotaxin-3

     

    8VETFGTTSYYDDVGLL23

    (all four sulfation states)

     

    Single sulfation enhances binding to

    CCL11, 24 and 26 3- to 30-fold.

    Sulfation of Y16 and Y17 gives different

    affinity enhancements.

    Double sulfation enhances affinity

    additively for CCL11 and CCL24,

    cooperatively for CCL26.

    Sulfopeptides bind to N-loop/β3 site.

    (Simpson et al., 2009;

    Zhuet al., 2011)

    CCR5

    CCL5/RANTES

    1NleDYQVSSPIYDINYYTSEPSQKINV25

    Nle = norleucine

    (non-sulfated; Y10/Y14-disulfated)

    Sulfation of Y10 and Y14 enhances affinity for CCL5 by >100-fold.

    Sulfopeptide binds to N-loop/β3 site.

    (Duma et al., 2007)

    CXCR4

    CXCL12/SDF-1

    GS1MEGISIYTSDNYTEEMGSGDYDSMKEPAFREENANFNK38

    (non-sulfated; Y21-sulfated; Y7/Y12/Y21-trisulfated)

     

    Single and triple sulfation enhances affinity

    3- and 30-fold respectively.

    Sulfopeptides stabilize dimeric CXCL12.

    NMR structures of complexes determined

    and sTyr binding sites identified.

    (Veldkamp et al., 2006;

    2008; Seibert et al.,

    2008)

     

    1Potentially sulfated Tyr residues are shown in bold; acidic residues are underlined.



    Reference

    http://www.ncbi.nlm.nih.gov/books/NBK56012/ Neuroproteomics


    Justin P Ludeman and Martin J Stone;The structural role of receptor tyrosine sulfation in chemokine recognition. British Journal of Pharmacology (2014) 171 1167–1179.

    Levi S. Simpson, John Z. Zhu, Theodore S. Widlanski, and Martin J. Stone;  Regulation of Chemokine Recognition by Site-Specific Tyrosine Sulfation of Receptor Peptides. Chem Biol. 2009 February 27; 16(2): 153–161. doi:10.1016/j.chembiol.2008.12.007.




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     Design Rules for Molecular Beacons

     

    Many innovative technologies and methods for sensitive and accurate genetic analysis have been developed during the last few years. The polymerase chain reaction (PCR) has made it possible to detect tiny amounts of DNA or RNA sequences in cells, tissue, or blood samples. The real-time polymerase chain reaction (RT-PCR) is the most commonly used method for this type of analysis. The implementation of fluorescent detection strategies in combination with sensitive instrumentation allows for the accurate quantification of nucleic acids.

    Since their introduction in 1996 molecular beacons have become widely used in the biosciences. Specific hybridization of complementary sequences in DNA is the basic mechanism for the identification of target genes. Stem-loop oligonucleotide probes have been developed for specific and selective target detection and are the key for a well-designed molecular beacon based assay.

     

    The principle of operation of molecular beacons is illustrated in figure 1.

     

     

    Figure1: Principle of operation of molecular beacons. A molecular beacon contains a fluorophore-quencher pair, sometimes also called a donor-acceptor pair, a loop region, and a stem region. The stem region contains two complementary sequences. The sequence in the loop region is complementary to the target sequence. If the target sequence is not presence the complementary sequence regions in the stem hybridize and bring the fluorophore and quencher in close contact. In this conformation the fluorescence of the molecule is quenched. Hence no fluorescent signal is detected. The beacon is extended when the probe sequence binds to the target and increased fluorescence occurs.  


    A molecular beacon is a fluorescent-labeled oligonucleotide, usually 25 to 35 nucleotides long. A typical molecular beacon can be divided into four parts:

    1.  Loop

    An 18 to 30 single-stranded sequence region complementary to the target sequence.

    2.  Stem

    Two short, 5 to 7 nucleotide residues long oligonucleotides complementary to each other. The stem is attached to both termini of the loop.

    3.  5’-Fluorophore 

    A fluorescent dye is covalently attached at the 5’-end.

    4.  3’-Quencher 

    A nonfluorescent quencher dye is covalently attached at the 3’- end.


    To establish a molecular beacon RT-PCR assay the following steps are necessary:


    1.  Target design.



    2.   Primer design.



    3.   Optimization of the amplification reaction conditions using SYBR Green.



    4.   Molecular beacon design.



    5.   Molecular beacon synthesis and characterization.

     

    Molecular beacons hybridize at the annealing temperature with the target sequence, the amplification product in PCR. They do not interfere with with primer annealing and extension. Correctly designed molecular beacons allow the detection of different targets in the same assay tube. Molecular beacon based real-time PCR can be used for a variety of studies including the detection of genomic DNA sequences, single nucleotide polymorphisms (SNPs), messenger ribonucleic acids (mRNA) expression levels, as well as pathogens. In addition, molecular beacons can also be used as intracellular probes for the detection of DNA and mRNA.

     

    Target Design



    1.  The source and of the template and the sequence of the primers and template
         will affect the efficiency of the PCR.

    2.  Design primer pairs that amplify a target region of 75 to 250 basepairs.

    3.  Avoid selecting a molecular beacon target sequence that forms strong secondary
         structures.

    4.  Analyze the selected sequences using a DNA folding program such as DNA mfold
         or UNAFold (
    http://www.bioinfo.rpi.edu/applications/mfold).


    Primer Design



    1.  Design primers with a 50 to 60 % GC content.

    2.  Target a Tm between 50 to 65 °C.

    3.  Avoid strong secondary structures. 

    4.  Avoid repeats og Gs or Cs longer than three (3) bases.


    5.  Check that the primers are not complementary to each other and avoid
         primer-dimers.



    6.  Place Gs and Cs on the ends of the primers. 

    7.  Avoid false priming by verifying the primers specificity. Us BLAST, the
         “
    Basic Local Alignment Search Tool” http://www.ncbi.nml.nih.gov/blast/,
         to do so.


    8.  Test primer sets using PCR and SYBR Green as a reporter molecule.


     Figure 2: Structure of SYBR Green I.

    SYBR Green I is a commonly used fluorescent DNA binding dye. The dye binds all double–stranded DNA and allows detection and monitoring by measuring the increase in fluorescence throughout the PCR cycle. The excitation and emission maxima of SYBR Green I are 494 nm and 521 nm, respectively.

     

    Molecular Beacon Design

     

    Molecular beacons should be designed such that they are able to hybridize to their targets at the annealing temperature of the PCR. However, the free molecular beacon should stay closed and be not fluorescent at this temperature. Proper design will achieve this. The Tm of the probe can be predicted with the help of the “percent-GC” rule. Several software packages are now available for this purpose.

     

    Usually a probe length of 22 to 30 nucleotides is used but can be expanded from 18 to 30 bases. The Tm of the probe should be 7 to 10 °C above the annealing temperature of the PCR. To test the designed beacon, thermal denaturation profiles can be performed. Longer loop sequences make the probe-target hybrids containing mismatches more stable at the annealing temperature of the PCR.


    1.  In the presence of a perfectly complementary target the molecular beacon
         must form a stable probe-target hybrid.



    2.  In the presence of a mismatched target the molecular beacon must
         remain closed.



    3.  Select a probe sequence that will dissociate from its target at temperatures 5 to
         8 °C higher than the annealing temperature of PCR.



    4.  Determine the window of discrimination, the range of temperature in which
         perfectly complementary probe targets can form and in which mismatched
         probe-target hybrids cannot form, by measuring the fluorescence of solutions
         of molecular beacons in the presence of each kind of target as a function of
         temperature. 

     

    STEM-LOOP FORMAT DESIGN

     

    After selecting the sequence for the probe, two complementary arm sequences need to be added on either side of the probe sequence. The stem should be stable at the annealing temperature of the PCR. PREMIER Biosoft offers a free design tool http://www.premierbiosoft.com/qpcr/ for this. A list of design guidelines are listed below.




    1.  The probe may contain some bases (usually some
    Gs and Cs) to form the 
         beginnings of a stem.



    2.  Divide the primer element from the stem by a C18 (also called HEG,
         hexethylene glycol) group.



    3.  Use fluorophores that can be added by phosphoramidite chemistry for
         optimum yields, although other labels can be attached through post-
         synthesis labeling chemistries.

    4.  Include a dark quencher adjacent to the spacer, at the end of the 5'-stem.

    5.  Avoid placing a
    G next to the fluorophore as this leads to lower fluorescence.



    6.  Design the stem to have a
    ΔG0 of about -1.5 to -2 kcal/mol. The stem strength
         will also depend upon the length of the intervening loop. Short loops give
         higher
    Tms.



    7.  Stems should be as short as possible, 5 or 6 bases are preferred (seven or
         more may lead to baseline drift).



    8.  Stems will be mostly GC. A standard stem: CCGCGC-loop-GCGCGG may be
         used but the exact
    Tm of this stem will vary depending upon the length of the
         probe element. 

    9.  
    Model the structuresof:

    9.a. The unincorporated Scorpion to confirm the
    Tm of the stem.



    9.b. The extended Scorpion (essentially the amplicon, plus the probe element).
           Ensure the correct strand of the amplicon is used: if the probe selected goes
           on the reverse primer, use the reverse-complement of the sequence, plus the
           forward strand probe. The Δ
    G0 of the second construct MUST be more negative
           than that of the stem although the
    Tm may be higher for the unincorporated
           version than the extended product.

     

    Fluorophore/quencher pairs

     

    The classic molecular beacon first presented by Kramer and colleagues was designed with an EDANS/Dabcyl fluorophore/quencher pair. Molecular beacon based assays containing this type of fluorophore/quencher pairs rely upon resonance energy transfer-mediated, intramolecular fluorescence quenching that occurs in the intact molecular beacon. Efficient fluorescence quenching is a result of favorable energetic overlap of the EDANS excited state and the absorption by DABCYL. The relatively long excited state lifetime of the EDANS fluorophore is also an additional key factor.

     

    Figure 3: Structure of the original molecular beaconused by Tyagi and Kramer in 1996. The molecular beacon consists of a 15 nucleotide long probe sequence. 

    This sequence is embedded between two complementary 5 nucleotide long arm or stem sequences. The fluorophore, EDANS (5-((2-Aminoethyl)-amino)-naphthalene-1-sulfonic acid) is conjugated to the 5’-terminal phosphate using a –(CH2)6-S-CH2-CO- linker. The quencher, DABSYL (4-(4-Dimethylaminophenylazo) benzenesulfonyl chloride, 4-(Dimethylamino) azobenzene-4′-sulfonyl chloride, DABS-Cl, Dabsyl chloride) is connected to the 39-terminal hydroxyl group using a –(CH2)7-NH- linker.

    Many more fluorophore/quencher pairs are now commercial available for the synthesis of molecular beacons. Table 1 list the quenching efficiency for different fluorophore-quencher combinations.

     

    Table 1:  Static quenching efficiency of different fluorophore-quencher combinations (Marras et al. 2002).

     

    Fluorophore

    Emax (nm)

    Dabcyl

    (Amax 475 nm)

    BHQ-1

    (Amax 534 nm)

    QSY-7

    (Amax 571 nm)

    BHQ-2

    (Amax 580 nm)

    Alexa 350

    441

    95 %

    97%

    97 %

    96 %

    Cy2

    507

    95 %

    98 %

    96 %

    97 %

    Alexa 488

    517

    94 %

    95 %

    95 %

    93 %

    FAM

    517

    91 %

    93 %

    93 %

    92 %

    Alexa 430

    535

    76 %

    92 %

    77 %

    96 %

    Alexa 532

    551

    93 %

    95 %

    96 %

    93 %

    Cy 3

    564

    94 %

    97 %

    95 %

    93 %

    Alexa 546

    570

    93 %

    98 %

    98 %

    96 %

    TMR

    577

    83 %

    87 %

    87 %

    86 %

    Cy 3.5

    593

    89 %

    96 %

    95 %

    95 %

    Alexa 568

    599

    91 %

    98 %

    99 %

    98 %

    Texas Red

    603

    96 %

    98 %

    98 %

    97 %

    Alexa 594

    612

    90 %

    95 %

    95 %

    94 %

    Alexa 633

    645

    96 %

    98 %

    97 %

    97 %

    Cy 5

    663

    84 %

    96 %

    79 %

    96 %

    Cy 5.5

    687

    82 %

    96 %

    74 %

    95 %

    Alexa 660

    690

    81 %

    96 %

    94 %

    95 %

    Alexa 680

    702

    81 %

    94 %

    90 %

    93 %

     

     

    Emax the emission maximum of the fluorophore, Amax absorption maximum of the quencher

     

    Reference

    Basic Local Alignment Search Tool - BLAST: http://www.ncbi.nml.nih.gov/blast/

    Beacon Designer 7.9: http://beacon-designer.software.informer.com/7.9/

    G. Goel, A. Kumar, A.K. Puniya, W. Chen and K. Singh; Molecular beacon: a multitask probe Journal of Applied Microbiology 2005, 99, 435–442.

    Jacqueline A. M. Vet, Salvatore A. E. Marras; Design and Optimization of Molecular Beacon Real-Time Polymerase Chain Reaction Assays. Oligonucleotide Synthesis, Volume 288 of the series Methods in Molecular Biology pp 273-290.

    Marras SA, Kramer FR, Tyagi S (2002) Efficiencies of fluorescence resonance energy transfer and contact-mediated quenching in oligonucleotide probes. Nucleic Acids Res 30(21):e122.

    mfold or UNAFold: http://www.bioinfo.rpi.edu/applications/mfold

    Molecular Beacons – Yang, C. J., and Tan, W.; Editors. Springer Heidelberg New York Dordrecht London. ISBN 978-642-39109-5.

    Molecular Beacons: www.molecular-beacons.org

    NUPACK– nucleic acid package:http://nupack.org/partition/new

    OligoCalc– SimGene.com:  http://www.simgene.com/OligoCalc

    PREMIER Biosoft offers a free design tool: http://www.premierbiosoft.com/qpcr/

    Primer 3– SimGene.com:  http://www.simgene.com/Primer3

    POLAND - thermal denaturation profiles of double-stranded RNA, DNA or RNA/DNA-hybrids: http://www.biophys.uni-duesseldorf.de/html/local/POLAND/poland.html

    Sfold– Wadsworth Center NYS: http://sfold.wadsworth.org/cgi-bin/index.pl

    Tyagi S, Kramer FR.; Molecular beacons: probes that fluoresce upon hybridization. Nat Biotechnol. 1996 Mar;14(3):303-8.


    Zipper H, Brunner H, Bernhagen J, Vitzthum F. Investigations on DNA intercalation and surface binding by SYBR Green I, its structure determination and methodological implications. Nucleic Acids Research. 2004;32(12):e103. doi:10.1093/nar/gnh101.


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  • 09/25/15--00:00: Light-sensitive nucleotides
  •  Light-sensitive nucleotides


    Light-sensitive oligonucleotides, also called Caged oligonucleotides, allow for the control of complex chemical and biological reactions through photoactivation using UV light. The use of light allows the controlled irradiation of biological samples, both spatially and temporally. To allow for optochemical regulation of DNA-based reactions, such as the deactivation of genes, in the last 20 years researchers have developed several different approaches that use photo-labile caging groups on nucleotides and oligonucleotides.


    Figure 1: Structure of NPE-caged ATP. The chemical structure and space filling model of adenosine-5’-triphosphate P3-(1-(2-nitrophenyl) ethyl) ester, usually provided as disodium salt (NPE-caged ATP), is illustrated here.


    The term “caging” refers to the chemical attachment of molecular groups such as the conjugation of a photo-labile protecting group to a biologically active molecule at a specific molecular location. Attachment of the caging groups renters the active biological molecules inactive. Irradiation with light at the required wavelength allows the selective removal of the photo-labile or caging group. The removing of the caging group reactivates the activities of the biological molecule studied.

    Caged nucleotides are nucleotide analogs in which the terminal phosphate contains a blocking group, usually conjugated via an ester bond that renders the molecule inactive. Ultraviolet photolysis of the caged nucleotide results in a rapid and localized release of the free nucleotide at the site of illumination.

    Since the last 20 years caged ATP, ADP , cAMP, GTP-γ-S have been available. These caged nucleotides have been used for the investigation of the molecular basis of skeletal fiber contraction. The mechanism of ATPases, other molecular motors, cellular receptors, ADP/ATP transport, intracellular release of cAMP, as well as G-protein coupled signaling pathways have been studied using photolysis. The structure for NPE-caged ATP is shown in figure 1. Photoactivation to start the uncaging reaction of these molecules is accomplished by exposing them to ultraviolet light at wavelengths ≤360 nm. Light sources useful for this task include lasers, flash-lamps, and suitably equipped fluorescence microscopes.

    DNA functions can be optochemically controlled via nucleobase-caging approaches.

    Different caging approaches for the regulation of oligonucleotide hybridization with light have been developed (Liu and Deiters, 2014). These are: 


    1.  Photo-deactivation via light–induced strand breakage.

    2.  Photo-activation via light-induced release of an inhibitor strand.

    3.  Photo-activation via linearixation of a light-cleavable circular oligonucleotide.


    4.  Photo-activation via photolysis of caged nucelobases.

    5.  Photo-deactivation via removal of nucleobase-caging groups from inhibitor strands.

    6.  Reversible caged-molecules.

    -.-


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    Caged nucleobases for optochemical control of DNA functions

    Caged molecular probes, or light-sensitive oligonucleotide based probes, enable the rapid release of biologically active molecules. Caging technology can provide results that cannot be obtained by other means. Adding photolabile caging groups into oligonucleotides has become a method or strategy to conditionally control oligonucleotide activities with the help of light. Hence, caged molecules are useful photolabile and light-sensitive molecular tools. Synthetic oligonucleotides can be designed as probes for the study of modulations of genes as well as to investigate gene functions.

     

    Usually, caging moieties are designed to interfere maximally with the binding and activity of target molecules, as well as their interactions with other molecules. Flash photolysis, the activation of the caged molecule, using light pulses at ≤360 nm, releases the active molecule as a pulse of the active compound. The uncaging reaction can be accomplished using UV light in a fluorescence microscope, with a UV laser or a UV flashlamp.

    Regulation of translation

     

    Regulation of translation is possible with the


    1.   use of light-activated antisense agents,
    2.   use of small interfering RNAs (siRNA),


    3.   use of antagomirs.


    Antagomirs are also known as anti-miRs or blockmirs. They are a class of engineered synthetic oligonucleotides that are complementary to specific microRNA (miRNA) target sequences and can be used to block this miRNA target and/or silence endogenous miRNA.

     

    Regulation of transcription


    Regulation of transcription is possible with the


    1.   use of caged triplex forming oligonucleotides,


    2.   use of DNA decoys, 
    3.   use of light-controlled aptamers.

    Reference

     

    Qingyang Liu and Alexander Deiters; Optochemical Control of Deoxyoligonucleotide Function Via a Nucleobase-Caging Approach. Acc Chem Res. 2014 January 21; 47(1): 45–55. doi:10.1021/ar400036a.



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     Chemical structures of caged nucleobases

     

    Most of the caging groups described in the literature employ the o-nitrobenzylic group. But other structural types have been reported as well. The caging or light-sensitive group can be synthetically incorporated into biologically active molecules such as amino acids, nucleotides, and oligonucleotides for the design of specific molecular probes. In general, for the conjugation reaction, a linkage to a hetero-atom is utilized. Common hetero atoms to which the caging moieties are linked to are oxygen (O), sulfur (S), or nitrogen (N) atoms. Functional groups used for the formation of the linkage can be ethers, thioethers, esters, including phosphate or thiophosphate esters, amines or similar groups. The structure of the caging moiety, as well as the atom to which it is linked to, affects the efficiency and the wavelength needed for the uncaging reaction. 



    Figure 1: 
    Structures of different caging groups.

    Most caging groups show good water solubility and are very fast to uncage with high quantum yields. Many of them release biologically inert photolytic by-products when uncaged. Typical uncaging rates are reported to be in the microseconds.

    Nomenclature for caging groups used here:

    CNB      =  α-carboxy-2-nitrobenzyl;
    NPE      =  1-(2-nitrophenyl)ethyl;
    DMNB   =  4,5-dimethoxy-2-nitrobenzyl;
    DMNPE  =  (4,5-dimethoxy-2-nitrophenyl)ethyl;
    CMNB   =  5-carboxymethoxy-2-nitrobenzyl.


    The addition of dithiothreitol (DTT) to caging experiments can help prevent the potential cytotoxic reaction between amines and 2-nitosobenzoyl by-products.

     

    The following is a list of caged nucleobases that have been incorporated into oligonucleotides. Light-sensitive caging groups are shown in red.


    A:  A photo-active C8 thioether-linked adenosine.

     

     

    Figure 1: Photo-active C8 thioether-linked adenosine. This group has been used to cage DNAzyme to control its enzymatic activity (Liu and Deiters 2014).

     

    Ting et al. in 2004 report the synthesis and photochemical properties of a this nucleoside: 8-(2-(4-imidazolyl)ethyl-1-thio)-2'-deoxyriboadenosine. Its light sensitivity was evaluated via an examination of the photoinduced reactivation of DNAzyme 8-17E from an inactive form that contained a single nucleotide modification. The photoinduced reversion of 8-(2-(4-imidazolyl)ethyl-1-thio)-2'-deoxyriboadenosine to unmodified deoxyadenosine restorated the activity of the DNAzyme.

     

    B:  1-(Ortho-nitrophenyl)-ethyl (NPE) photolabile group

     

     The 1-(ortho-nitrophenyl)-ethyl (NPE) photolabile group has been used early onto to design and synthesize a photolabile chelator, nitrophenyl-EGTA (NPE-EGTA), that can selectively bind calcium ions (Ca2+) with high affinity. Photolysis of the chelator rapidly releases the calcium ions. The rapid controlled and localized increase in calcium ion concentrations has enabled studies of kinetics, regulatory and structural mechanisms of calcium based intracellular communications. More recently the NPE group has been applied to five nucleobases.



    Figure 2:  
    Structures of NPE labeled nucleobases. The NPE group has been used for the study of biological processes since the 1980s. Nucleobases in which this caging group have been incorporated are shown (Liu and Deiters 2014).

     

    C:   2-(Ortho-nitrophenyl)propyl (NPP)

     

    This caging group has also been applied to nucleobases. Over the years different designs were used and tested with the goal to improve stability and quantum yields of the caging group during decaging reactions.



    Figure 3:  
    Structures of NPP labeled nucleobases. The NPP caging group has been used for the nucleobases depicted here (Liu and Deiters 2014).

     

    D:   6-Nitropiperonyl methyl group (NPM) and hydroxymethylene analog
           (NPOM).


    This caging group was developed to improve decaging rates. The 6-nitropiperonyl-methyl group was conjugated to nucleobases to red-shift the absorption maximum and the decaging wavelength. In addition, the stability of the caging group was also improved during oligonucleotide synthesis. However, for almost all caging groups, the decaging wavelength falls typically between 360 and 366 nm. These caging groups are reported to be stable under ambient light.




    Figure 4:
    Structures of NPM and NPOM labeled nucleobases. The NPM caging group has recently been used for cytosine and NPOM for guanine, thymine and uracil (Liu and Deiters 2014).

     

    E:   Diethylaminocoumarin (DEACM) and nitrobenzofuran (NDBF).

     

    Coumarin based caging groups have also been investigated for their use in nucleobases. Since they require a leaving group with a low pKa their use is limited.The diethylaminocoumarin (DEACM) group was used for the caging of guanine. The nitrobenzofuran (NDBF) group was also employed for the caging of several nucelobases. However, this group may not be as versatile as the caging groups reported earlier.



    Figure 5:
      Structures of DEACM and NDBF labeled nucleobases. The coumarin based caging group enables photolysis at >405 nm (Liu and Deiters 2014).

     

    F:  o-Nitrobenzyl phosphate ester based caging

     

    Dussy et al. in 2002 used an o-nitrobenzyl ester based caging group to enable phototriggered bond cleavage.

     

    Figure 6: Structure of an o-nitrobenzyl ester based caging DNA building block.

     

    The research group synthesized photocleavable nucleotides based on the photochemistry of o-nitrobenzyl esters through the introduction of o-nitrophenyl groups at the 5′C position into the oligonucleoside sugar backbone. These modified nucleosides are able to build stable DNA duplexes. Oligonucleotides modified this way can be cleaved site-specifically by irradiation with >360 nm light with high efficiency.

     

    Reference


    Adrian Dussy, Christoph Meyer, Edith Quennet, Thomas A. Bickle, Bernd Giese and Andreas Marx; New Light-Sensitive Nucleosides for Caged DNA Strand Breaks. Volume 3, Issue 1, pages 54–60, January 4, 2002.

     

    G C Ellis-Davies, J H Kaplan; Nitrophenyl-EGTA, a photolabile chelator that selectively binds Ca2+ with high affinity and releases it rapidly upon photolysis. Proc Natl Acad Sci U S A. 1994 January 4; 91(1): 187–191. PMCID: PMC42911.

     

    Ting R, Lermer L, Perrin DM. Triggering DNAzymes with light: A photoactive C8 thioetherlinked adenosine. J Am Chem Soc. 2004; 126:12720–12721. [PubMed: 15469235].



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     A General Design for Caging Groups

     

    To improve caging groups for the use of different applications such as photolithography or DNA arrays several types of caging groups have been designed. Figure 1 illustrated a general chemical structure for a variety of approaches used for the design of caged molecules.

     

    Figure 1: Chemical structure for the general design of caging groups.

     

    Y = amino acids, XNH-, or C51 oxygen on DNA or RNA,

    R1 and R2 = Hydrogen, alkyl, aryl, benzyl, halogen, hydroxyl, alkoxyl, thiol, thioether, amino, nitro, carboxyl, formate, formido, sulfide, phosphidogroup,

    R3 = alkoxy, aryl, alkenyl, hydrogen group.

     

    {Source: Patent WO 1992010092 A1; 1992}.

     

    The perturbation effect for each caging group can vary based on oligonucleotide function, the number of caged nucleobases used per oligonucleotide, as well as the position of the caging group within the oligonucleotide probe. However, often a single caging group placed at a crucial site is sufficient for blocking activities, for example, to interfere with or study protein interactions or catalytic DNAzyme activities. Multiple caging groups are often required for the regulation of base-pairing interactions between oligonucleotides. Studies have shown that in general one caging group placed every 5 to 6 bases and evenly spaced throughout an oligonucleotide can fully abrogate or block hybridization to the complement. Removing the caging groups through irradiation will allow restoring hybridization.

     

    To conclude, well designed photo-caged oligonucleotides or probes can be used for the study of biological processes via light- or optochemical-regulation. However, recent scientific reports indicate that the scope of application for this technology is much wider. Caged oligonucleotides may enable new types of applications in DNA based computation, DNA/RNA sensing, as well as DNA nanotechnologies. The future may see their use for the study of gene functions, effects of non-coding RNA, in embryo development, cell motility, and other research areas. Also, caging groups can be combined with other modified nucleobases such as bridged nucleic acids (BNAs) to design new types of oligonucleotide based probes and tools.


    Reference

     

    Adrian Dussy, Christoph Meyer, Edith Quennet, Thomas A. Bickle, Bernd Giese and Andreas Marx; New Light-Sensitive Nucleosides for Caged DNA Strand Breaks. Volume 3, Issue 1, pages 54–60, January 4, 2002.

     

    G C Ellis-Davies, J H Kaplan; Nitrophenyl-EGTA, a photolabile chelator that selectively binds Ca2+ with high affinity and releases it rapidly upon photolysis. Proc Natl Acad Sci U S A. 1994 January 4; 91(1): 187–191. PMCID: PMC42911.

     

    Ting R, Lermer L, Perrin DM. Triggering DNAzymes with light: A photoactive C8 thioetherlinked adenosine. J Am Chem Soc. 2004; 126:12720–12721. [PubMed: 15469235].

     

     


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    Molecular Diagnostics and the Detection of DNA and RNA Viruses

    Many molecular diagnostics techniques or tests first developed for their use in molecular biology and biotechnology laboratories are now routinely used in clinical laboratories. Molecular virology refers to the specific detection of viral infection utilizing molecular detection methods. However, to allow for transparent quantitative molecular testing and data sharing between laboratories and institutions DNA and RNA based reference materials or standards are needed. The benefit of the availability of reference materials or standards for quantitative molecular testing is now been recognized internationally. C
    linical laboratories, independent control and reference material manufacturers, in vitro diagnostics (IVD) manufacturers, and regulatory and policy makers, such as the AACC and FDA, are involved in developing standardized materials and protocols. Molecular probes or primers designed using natural or artificial nucleic acids are enabling key tools for the development of sensitive molecular diagnostics assays. For example, recently, bridged nucleic acids (BNAs) have been used to allow for the quantitative and sensitive detection of different mutant alleles.


    Figure 1: 
    3D Model of RNA and DNA structures. (Source: RCSB Protein Data Bank (PDB) DNA and RNA structural data:
     

    Since Watson, Crick and Wilkens received the Nobel Prize in Physiology or Medicine in 1962 for solving the DNA double-helix structure, many new nucleic acid-based diagnostic tools or assays have been developed that allow analysis of DNA and RNA molecules. Over time, they have been given many different names, such as Molecular Diagnostics, Molecular Pathology, Molecular Genetics, Molecular Genetic Pathology, Molecular Hematology-Oncology, Molecular Oncology, Molecular Virology, and Molecular Microbiology. However, the term “Molecular Diagnostics” covers them all. Many of these molecular diagnostics techniques have migrated to clinical laboratories. In the early years of biotechnology, in the 1980s to the 1990s, Southern blot hybridization was the technique used the most. Since it was a time-consuming, labor-intensive, and less sensitive diagnostic method, centers of excellence performed this test exclusively. The polymerase chain reaction (PCR) invented in the 1980s, an in-vitro nucleic acid amplification technique, has now become the dominant method after several rounds of optimization. In molecular virology, molecular testing is now possible in a short period-of-time, often taking just 20 to 30 minutes. In the case of HIV/AIDS, viral load testing is now possible as well.




    Figure 2:
    3D Model of the H1N1 Influenza Virus. (Source: Center for Disease Control and Prevention: www.cdc.gov, http://www.cdc.gov/h1n1flu/images.htm)

    As many people have experienced first hand, during the flu season, many people can get infected with the flu virus. However, other viruses can infect people in many different ways. Viral infections can happen by inhaling or swallowing them, by being bitten by an insect, or through sexual contact. Examples of viruses that cause a disease are the Ebola virus, human papillomavirus (HPV), hepatitis C virus (HPC), hepatitis D virus (HPD), human immunodeficiency virus (HIV/AIDS), dengue fever virus, and many others. Often, viral infections target the nose, throat, and upper airways. Diagnosis of the infection type is based on symptoms, blood test, and cultures, as well as the examination of infected tissues. Common viral infections like measles, rubella, or chickenpox are often just diagnosed based on symptoms. However, the use of more precise tests such as blood tests and cultures offer a more accurate diagnosis.

    The polymerase chain reaction or PCR allows making many copies of the viral genetic material. The PCR method enables the rapid and accurate identification of a particular virus. Therefore, PCR-based molecular detection of a virus is currently the method of choice for its correct identification.

    In the last decade, the molecular diagnostics field has been growing rapidly. Tests or assays used in molecular diagnostics detected specific DNA or RNA sequences. Specific molecular probes and primers are designed for this purpose. Molecular diagnostics tests or assays are now commonly used for the diagnosis of a disease. Furthermore, these assay types can be used to monitor or detect the risk for a disease, as well as to help decide which therapies will work best for infected patients. Furthermore, molecular diagnostics now enables personalized medicine.

    Many innovated assays and instruments have been developed in the last decades enabling in vitro diagnostics (IVD). Examples are real-time PCR based instruments such as thermal cyclers, genetic analyzers, Sanger sequencing and next-generation sequencing.

     

    What is a virus?


    A virus, in biology, is a small infectious agent that can only replicate inside living cells of other organisms. Viruses are ultramicroscopic, metabolically inert, agents that can infect all kinds of species, such as animals, plants, and microorganisms, including bacteria, single-celled microorganism (archaea), and humans. Viruses typically consist of a nucleic acid molecule, DNA or RNA, within a protein coat, or sometimes a surrounding envelope. Viruses are quite small and tiny. Therefore, they can not be observed in a light microscopy. Viruses are only able to multiply within living cells of a host. Diseases caused by a virus are known as a “viral infections.” 

     

    Types of Molecular Diagnostics Assays

     

    A typical molecular diagnostic assay requires the following three basic steps:

    (1)   Extraction and purification of DNA or RNA.

    (2)   Amplification or copying targeted nucleic acid sequences. Often fluorescent dyes
           are also attached to the amplified oligonucleotides during this step.

    (3)   Detection of amplified target sequences using real-time PCR, microarrays,
           multiplexing, or sequencing.

      

    The following categories of molecular assays are available for detection of a viral infection in tissue and exfoliated cell samples. The basis for the tests is the detection of DNA or RNA sequences:

     

    (1)   Assays using Southern Blotting or Southern Transfer Hybridization, (STH),
           Dot Blot Hybridization (DB), and In Situ Hybridization (ISH).

    (2)   Signal amplified hybridization assays, for example, a hybrid capture assay.

    (3)   Target amplification assays. For example, PCR and in situ PCR.

     

    Southern blot hybridization and in situ hybridization


    Originally Southern blot hybridization (STH) and in situ hybridization (ISH) were used and can still be used. Unfortunately, these techniques often have serious shortcomings. Southern blot hybridization assays require large amounts of DNA and are laborious tasks, and are often not reproducible. On the other hand, in situ hybridization or ISH base assays often show only moderate sensitivity for the virus tested.


     

    PCR and in situ PCR


    PCR and in situ PCR, techniques that amplify the target sequences, are extremely sensitive and specific. Using this method, viral DNA or RNA is amplified in vitro by a DNA polymerase or RNA polymerase to generate adequate amounts of target nucleic acids. Amplified nucleic acids are then either directly visualized on gels, or detected by specific hybridization probes.

    Theoretically, PCR can detect one copy of a target sequence in a given sample. However, in practice, a PCR-based method can detect approximately 10 to 100 viral genomes in a background of 100 ng cellular DNA. Using PCR, small amounts of DNA in the range of 10 to 100 ngs can be detected. PCR is, therefore, the ideal method for the analysis of specimens with low DNA or RNA content. However, proper sample collection and handling is essential to achieve maximal sensitivity. Brush-sampling devices have been shown to be optimal for sample collection.

    Most assays allow differentiating between high and low-risk virus infections. However, individual virus types cannot be identified without additional more specific tests or assays.


    Examples of testing types or assays


    Examples of testing types or assays used for virus detection are:

    (1)   Detection of proviral DNA by real-time PCR.

    (2)   Detection of viral infection and load using branched DNA (bDNA) tests.

    (3)   Detection of viral load by nucleic acid sequence-based amplification (NASBA)
            tests.

    (4)   Detection of viral vectors such as adenovirus, MVA, AAV, and others, by
            quantitative PCR and RT-PCR.

    (5)   Detection of cytokines and other biomarker responses.

    (6)   Detection of host restriction factors.

    (7)   Examination of virus-host interactions and global gene expression profiles.
           These types of tests help to understand molecular mechanisms of immune
           response and protection.

    (8)   Virus load determination.

     -.-

    Control Templates for Molecular DNA and RNA Diagnotics 

    Bio-Synthesis can help you synthesizing your synthetic standards and standardized material according your specifications. Bio-Synthesis has been successful using chemical and enzymatic synthesis for long DNAs and RNAs of up to 400bases. Oligonucleotides between 80-400 bases are synthesized using the latest nucleic acid technology and purified using DNAse and RNase free PAGE electrophoresis combined with HPLC. Our oligonucleotide synthesis services include mass spectrometry analysis. Purified oligonucleotides undergo stringent analytical HPLC or gel electrophoresis to determine final purity.

    -.-

    Reference

    American Association for Clinical Chemistry (AACC) (https://www.aacc.org/store/cds/10100/viral-load-testing-diagnostic-principles-and-clinical-practice---cd).

     

    U.S. Food and Drug Administration (FDA) – Guidelines for Standardized Assays and Protocols: http://www.fda.gov/medicaldevices/deviceregulationandguidance/guidancedocuments/ucm180306.htm

     

    Center for Disease Control and Prevention:

     

    http://www.cdc.gov/flu/images.htm

     

    http://www.cdc.gov/STD/HPV/STDFact-HPV.htm

     

    http://www.cdc.gov/vhf/virus-families/filoviridae.html

     

    Info on currently approved molecular tests at the Association of Molecular Pathology website: http://www.amp.org/

     

    Roberta M. Madej, Jack Davis, Marcia J. Holden,Stan Kwang, Emmanuel Labourier, and George J. Schneider; Review; International Standards and Reference Materials for Quantitative Molecular Infectious Disease Testing. Journal of Molecular Diagnostics, , Vol. 12, No. 2, March 2010

     

    Merckmanuals: http://www.merckmanuals.com/home/infections/viral-infections/overview-of-viral-infections

     

    http://www.pathology.washington.edu/clinical/women/detection/

     

     


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    Bioconjugate Chemistry for Molecular Engineering


    Recent developments in protein engineering, also known as bioengineering, have enabled the production of modified biomolecules such as proteins, DNA and RNA oligonucleotides containing desired properties useful for the creation of novel applications and nano-devices. During the last decades, chemical conjugation reactions have been applied in biology, molecular biology, and molecular medicine for various applications.

    A "bioconjugate" refers to a molecular species that can be produced by living systems or synthetically by chemists or biologists. A bioconjugate is composed of at least two different molecular parts of biological origin. Bioconjugates used in organisms or cells are designed such that they are water soluble or that cells are able to compartmentalize them. For synthetic production of bioconjugates different bioconjugation chemistries are used.

    Bioconjugate chemistry, often also called bioconjugation chemistry, is a research field that studies the linking of one molecule to another by chemical or biological means. Typically, the resulting complexes are formed from at least one biomolecule, however, several molecules may be conjugated together as well. In addition, purely synthetic conjugated molecules are possible as well.  

    The study and use of chemical conjugation reactions, now also known as bioconjugation reactions, has more recently evolved into an important research topic as can be monitored by the number of publications in this field. For example, site-specific bioconjugations of a multitude of biomolecules to proteins, DNA, RNA, and carbohydrates, or to each other, have been developed. The resulting conjugates are useful for applications such as ligand discovery, disease diagnosis, and high-throughput screening, in vivo imaging, sensing, catalysis, therapeutics, as well as cell targeting. More recently, polymer brushes linked with biotin moieties allowing for the development of streptavidin-mediated conjugation capture agents in NanoVelcro chips have been engineered. These conjugates are a new type of molecular probes for prenatal diagnostics (GEN July 2015).  




    General bioconjugation chemistry schemes are illustrated in f
    igure 1. Typically, bioconjugation reactions are employed to couple biomolecules to surfaces or solid supports. Typical supports are beads, gold surfaces, nitrocellulose or dextran based arrays. The use of these chemistries allows the synthesis of oligonucleotide-, peptide-, or protein based libraries coupled to a solid support such as a micro-chip.  Typical applications are screening or sequencing by hybridization. The conjugation of oligo-nucleotides to a support is shown as an example. However, peptides, carbohydrates and proteins can also be used in a similar fashion. Other nanostructures such as dendrimers, cyclodextrin or cellulose, modified or unmodified, are also often utilized as spacers or amplifying moieties, usually between the support and the biomolecule selected for conjugation. 



    Figure 1: General schematic of bioconjugation chemistries. The conjugation of oligonucleotides to a solid support or surface is illustrated here. Three bioconjugation chemistries commonly used are reviewed. Carbonyl-diimidazole (CDI) activation to silanol groups is shown to the left. The surface functionalization technique using glycidooxypropyl-trimethoxy-silane (GOPS) is shown in the middle. And, the surface functionalization technique using 3-aminopropyltrimethoxysilane (APTMS) is shown at the right. Dendrimers or other branched polymers or other spacer molecules can also be used, often between the support and the molecule to be conjugated. 


    Bioengineering or Biological engineering is a new scientific field that applies engineering principles to biological systems. Broadly viewed, bioengineering can include elements of electrical, mechanical, computer science, materials, chemistry, biology and medical biology. Its main goal is to apply concepts and methods observed in biological systems to solve real-world problems in life sciences. Often medical biology is part of this endeavor. Bioengineering uses primarily knowledge gained from the fast developing field called “molecular biology.” A rapid development of many innovative techniques and methodologies pertinent to biological or medical applications using bioengineering principles leading to new applications in medicine, agriculture, and energy or electronic production occurred in recent decades. A new branch known as “biomimetics” strives to engineer new materials to mimic structures and functions of molecules found in living organisms using DNA, RNA, and protein molecules. The goal is the production or manufacture of new types of nano-materials, such as hydrogels,nano-particles, artificial proteins, antibodies, peptides, or dendrimers, and many others. However, the success of each conjugation reaction depends highly on its chemoselectivity under physiological conditions. Therefore, various chemoselective cross-linkers have been developed for the purposes of labeling and conjugation of selected molecules to functional groups available on target molecules. The following paragraphs describe several of these bioconjugation chemistries.

     

    (1)  Formation of amide bonds, including urea and thiourea moieties.

    Typically amide bonds are formed through the reaction of an amino group with a carboxylic acid. Activator reagents are utilized to form the bonds more efficiently. Activator molecules such as 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), carbonyl-diimidazole (CDI) or similar compounds are commonly used as illustrated in figure 2A. Figures 2B and 2C show the reaction of the amino group with active esters. The reaction of amino groups with isocyanate or isothiocyanate produces a urea or thiourea backbone, respectively, as depicted in figure 1D. Many biomolecules including proteins have many amino and carboxyl groups at their molecular surface.  Also, amino and carboxyl groups can be introduced to synthetic oligonucleotides and peptides with ease. Therefore, these chemistries are most widely used for the production of bioconjugates. Even some newly developed cross-linkers, for example, copper-free click chemistry based cross-linkers, use these types of reaction known to organic chemists for a long time, to modify biomolecules. Under standard conditions, these chemical reactions are normally quick and selective, forming stable amide bonds within a few hours. However, the majority of conjugation reactions are normally performed in neutral or slightly basic conditions, free of primary and secondary amines. EDC-mediated conjugations are an exception. In this case, reactions are best performed at slightly acidic conditions. Amide forming reactions are normally selective towards amino groups and are thermodynamically or kinetically controlled. However, isocyanates and isothiocyanates also react with the hydroxyl group to form carbamate and thiocarbamate bonds, respectively. Thus, these cross-linkers are also used to conjugate hydroxyl-containing molecules.


    Figure 2. Amide formation reactions frequently used in bioconjugation.


    (2)  Formation of thioethers

    The formation of thioesters is a widely used conjugation chemistry known for quite some time now. Reactions are very selective and specific since thiol groups are available in proteins that contain cysteine.  Some proteins, such as members of the C3/α-2M thioester protein family, and peptides that contain lanthionine, contain natural interchain thioester bonds. The formation of thiols bonds in biomolecules is relative straight forward. Also, incorporation of thiol groups into synthetic oligonucleotides and peptides is done with the help of standard chemistries. An advantage of the thioester chemistry is that primary amines do not interfere with the reaction avoiding the formation of byproducts. Also, reactions can be performed at a broad pH range, ranging from pH 2 to 10. Therefore, many hetero-bifunctional cross-linkers are typically designed to contain one thiol-reactive group andoneamino-reactive group. Reactive compounds that form thioether bonds or linkages include thiol-maleimide (Figure 3A), and thiol-haloacetate (Figure 3B). The lesser electronegativity between the sulfur and hydrogen atoms, compared to the oxygen and hydrogen atoms, make the thiol group less polar than the hydroxyl group. The reductive dealkylation of thioethers generates thiols.

     

     Figure 3. Frequently used bioconjugation thioether formation reactions.


    (3)  Conjugation reactions involving carbonyl group

    The presence of carbonyl groups in biomolecules is very limited. However, these are important functional groups allowing the conjugation of saccharide moieties. Except for the aldehyde and ketone groups of the linear saccharides, hydroxyl groups at the anomeric position of the cyclic saccharides normally are not reactive enough and, therefore, are not available for bioconjugation reactions. In particular, 1,2-diol groups in saccharides can be specifically oxidized to aldehydes with the help of sodium periodate.  Carbonyl groups can be introduced to biomolecules using cross-linkers such as N-succinimidyl-4-formylbenzamide (S-4FB).  Carbonyl groups are reactive towards primary amines, including hydrazine and oxyamine groups, whereas thiol and hydroxyl groups usually don’t interfere with the reaction. The reaction of a carboxyl group with a primary amine, hydrazine or oxyamine form Schiff bases, hydrazone or oxime moieties, respectively, as illustrated in figure 4. Oxime groups are quite stable and exist as two stereoisomers. However, Schiff bases and hydrazones are more labile, especially in acidic solutions or conditions. Therefore, the Schiff base is typically reduced to a secondary amine with the help of sodium cyanoborohydride, called reductive amination, as depicted in figure 4A. Furthermore, hydrazones can also be reduced and are typically stabilized by adding an aromatic group or groups to the resulting compounds in proximity to the hydrazone group.

     

    Figure 4. Conjugation reactions involving carbonyl groups.

     

    (4)  Thiol-exchange reactions

    Thiol-exchange reactions are widely used because these reactions produce cleavable conjugates.  Disulfide bonds are created between the conjugated molecules. Disulfide bonds containing conjugates can be cleaved via reduction by thiol reductases in tissue or cells. Furthermore, disulfide bonds can also be chemically cleaved in vitro using reducing agents such as mercaptoethanol (2-ME), tris(2-carboethyl)phosphine (TCEP), or dithiothreitol (DTT). However, the reactions for the formation of conjugates are usually slow but very selective towards the thiolated molecules. Amino or hydroxyl groups don’t usually interfere to produce byproducts. Although sometimes methanethiosulfonate reagents are used, 2-pyridyldithiol reagents are the most widely used thio-exchange cross-linkers, illustrated in figure 5. 



    Figure 5. The thiol-exchange reaction of 2-pyridyldithiol based cross-linkers.


    (5)  Click chemistry and tetrazine ligation

    Click chemistry has gained a huge momentum in bioconjugation due the development of the reagents recently.  Traditionally, click chemistry refers to the reaction of an alkynyl compound with an azido compound by the catalysis of Cu(I) (Figure 6A). Although this reaction is very selective and clean, coordination of Cu(I) with many ligands makes the complete removal from the conjugate problematic. Furthermore, the toxicity of copper limits the application of click chemistry in many conjugation chemistries.  Copper-free click chemistry overcomes this problem. Here, cyclooctyne is used instead of common alkynes, the tension in the 8-member ring promotes the click reaction,  no longer requiring Cu(I) catalysis.  Also, since recently needed reagents became available at a lower price, the copper-free click chemistry became very popular in recent years. Frequently-used cyclooctynyl reagents include the cross-linkers dibenzocyclooctynyl (DBCO) and bicyclo[6.1.0]nonynyl (BCN).  In addition, the Diels-Alder reaction and reverse Diels-Alder reaction has been studied for years as a conjugation chemistry. A recent successful development is the conjugation reaction of trans-cyclooctene to tetrazines, called, a tetrazine ligation (Figure 6B). Here, the tension in the trans-cyclooctene ring allows the reaction to occur at room temperature, relatively fast and without the need for a catalyst. 


    Figure 6. Click  chemistry (A) and tetrazine ligation (B)


    (6)  Photoreactive cross-linkers

    Photoreactions are only frequently performed in bioconjugation reactions. However, they are occasionally used for in-vivo or in-vitro crosslinking reactions.  Photoreactive cross-linkers commonly used are azidophenyl or diazirine compounds. Upon exposure to ultraviolet light, these compounds are activated and react with electron-donating groups, most frequently amino group, to produce conjugates (Figure 5). The reactions are normally not very fast and unselective, and may have multiple pathways. Also, products are not very clean. One advantage of photocrosslinking is that biomolecules that are selected to be conjugated can be mixed prior to the conjugation reaction, and the start of the reaction can be controlled.

    Figure 7.Photoreactions frequently used in bioconjugation.


    References

    Advances in Bioconjugation:   http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2901115/

    BC Bioconjugate Chemistry Journal:   
    http://pubs.acs.org/journal/bcches

    Chemical modifications and bioconjugate reactions of nanomaterials for sensing, imaging, drug delivery and therapy                                    






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    Design Guidelines for BNA based Oligonucleotide Probes
     

    Artificially modified nucleic acids, such as bridged nucleic acids (BNAs), can be used to increase the thermal stability of probes and primers while maintaining target recognition. Bridged nucleic acids contain key features that allow for this to happen. Rahman et al. in 2005 synthesized and determined the properties of 2’,4’-BNANC molecules. These nucleotide analogs contain an N-O bridged structure which favors sequence selective duplex and triplex formation in BNA/DNA chimeras. Reported duplex and triplex-forming abilities were slightly higher or similar to 2’4’-BNAs. However, observed nuclease resistance was as high as that of S-oligonucleotides.

    Figure 1: Design scheme for the synthesis of artificial nucleotide 2'4'-BNANC

    Bridged nucleic acids (BNAs) are artificial bicyclic oligonucleotides that contain a five-membered or six-memberedbridged structure with a “fixed” C3’-endo sugar puckering that is synthetically incorporated at the 2’, 4’-position of the ribose to afford a 2’, 4’-BNA monomer. BNA monomers can be incorporated into oligonucleotide polymeric structures using standard phosphoramidite chemistry. BNAs are structurally rigid oligonucleotides with increased binding affinities and stability. Oligonucleotide modifications are characterized by the presence of one or more bicyclic ribose analogs. The structural similarity to native nucleic acids and the presence of a nitrogen atom within the bicyclic ring leads to very good solubility in water and allows for easy handling of synthetic primers and probes. In contrast to peptide nucleic acids (PNAs) and minor groove binders (MGBs), but similar to LNAs, BNA monomers can be used for both primers and probes in real time quantitative polymerase chain reaction (RT-Q-PCR) assays.

    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. The thermal stability is depending on the number of BNA monomers present in the sequence, and BNA modifications greatly increase the melting temperature of oligonucleotides. Differences in melting temperatures (Tm) between perfectly and imperfectly matched nucleic acid duplexes allow for the discrimination even of single base mutations.

    DNA/DNA hybridization based molecular biology techniques rely on the accurate prediction of the melting temperature Tm. Many computer programs using different methods and parameters are available for the theoretical estimation of the experimental Tm value for short oligonucleotide sequences. However, often different values in the estimation of Tm are obtained when using different software packages.
     

    Limitations of Tm estimation calculations are well described in the literature. Ultimately the experimental measurement of Tm values is needed.

    BSI now offers the experimental determinations of Tm values for probes and primers as a service.


    Panjkovich and Melo, in 2005, compared several different melting temperature calculation methods for short DNA sequences and observed significant differences for the Tm values of short DNA oligonucleotides calculated by different Tm prediction methods. Their results indicate that care needs to be taken when estimating Tm values for oligonucleotides to avoid the failure of PCR experiments.

    In the same year, Chavali et al. performed a similar comparative analysis for the prediction of the melting temperature (Tm) for a large set of oligonucleotide sequences. The Tm was predicted using 25 oligonucleotide property calculators. Significant differences in Tm predictions for short DNA sequences were observed when a large number of sequences were tested. Of the 11 primer designing tools evaluated, Primer 3 and WebPrimer performed the best for the AT-rich templates, Exon Primer for AT = GC templates, and Primer Design Assistant, Primer3 and Primer Quest for GC-rich templates.

     

    Conclusion: Many Tm estimation calculation methods do not predict the Tm accurately. Ultimately the experimental measurement of Tm values is needed.

    Probe and Primer Design


    Tm Optimization Guidelines

     

    Guidelines to help increase the success of practical molecular biology applications when using BNAs, as modeled after Panjkovich and Melo, are listed as follows:

    (1)  Consider restrictions or limitations that each method has: For example, avoid
           sequences that form stable alternative secondary structures because such
           sequences do not follow a two-state transition;

    (2)  
    If possible, use oligonucleotide sequences that fall in the middle range of
           CG-content and are shorter 
    than 20–22mers. This is where most of the current
           Tm prediction methods agree;

    (3)   Avoid the use of sequences that fall in those regions of oligonucleotide feature space
            where none of the current methods agrees (see Panjkovich and Melo, 2005);

    (4)   For large-scale applications with sequences where a two-state transition is not known
            to occur, use consensus Tm calculation method as published by SantaLucia et
    al.,
           in 1996.

     

    Tm calculations

     

    Melting temperatures can be calculated using the nearest-neighbor model and thermodynamic data as described by SantaLucia et al. (1996).

     

    The equation used is as follows:




    Sums of enthalpy (ΔHd) and entropy (ΔSd) are calculated over all internal nearest-neighbor doublets.


    ΔSself is the entropic penalty for self complementary sequences, and


    ΔHi and ΔSi are the sums of initiation enthalpies and entropies, respectively.


    R
    is the gas constant (fixed at 1.987 cal/K·mol),


    C
    T is the total strand concentration in molar units and


    T
    m is the melting temperature given in Kelvin units.


    Constant b adopts the value of 4 for non-self-complementary sequences or equal to 1 for duplexes of self-complementary strands or for duplexes when one of the strands is in significant excess.

     

    The thermodynamic calculations assume that the annealing occurs in a buffered solution at pH near 7.0 and that a two-state transition occurs.

     

    Template preparation

     

    Proper sample handling and preparation is needed for every polymerase chain reaction (PCR) method used to monitor gene expression, for quantifying food-borne pathogens, testing viral load, or for any pathogen testing, including forensic analysis and clinical diagnostics. Inhibitors present in the original samples can reduce or even block DNA amplification. Proper pre-PCR processing is needed to avoid PCR inhibition during all steps of the PCR chemistry. To achieve optimal detection limits, high specificity, and short time-to-results, many sample preparation methods have been published in recent years. Many of them combine several sample preparation methods. To achieve detection of very low concentrations of food-borne pathogens, enrichment methods are required. 

    Sample preparation methods used for PCR can be divided into four major categories, but combination of these methods may also be needed for the detection of low concentration of target sequences:

     

    (1)      Biochemical methods

    For example, the extraction or purification of RNA or DNA with organic solvent such as phenol-chloroform. DNA and RNA extractions methods have now been automated for many samples. Often, cationic magnetic beads or silica-based filters or suspensions are used to separate nucleic acids from sample matrices.

     

    (2)      Culture enrichment methods

    These methods involve the cultivation of the target microorganism prior to PCR. The aim is to provide detectable concentrations of target cells prior to PCR.

    (3)     Immunological methods 

    Many of these methods are based on magnetic beads coated with antibodies to allow the separation of target cells from their natural environment and to concentrate them further. Immunomagnetic separation methods (IMS) can be used for both, DNA and RNA sample preparation. These have also been automated in recent years. Unfortunately, complex matrices can interfere with immune-capture method and may require further processing such as lysis and additional washing steps, or sometimes even an additional purification step prior to detection.

     

    (4)      Physical methods

    Examples of physical methods are aqueous two-phase systems, buoyant density centrifugation, flotation, centrifugation or ultracentrifugation, as well as filtration. The success of these methods depends on the physical properties of the target samples.

     

    (5)      Combination of methods 

    The combination of different methods may be needed or can be used to achieve optimal detection. However, combining different methods can be time-consuming and costly. In addition, the KIS (keep it simple) rule should always be applied.

     

    Hydrolysis Probes

     

    A hybridization probe consists of a fragment of DNA or RNA of variable length. In general, hybridization probes are between 100 to 1,000 bases in size. Originally hybridization probes were radioactively labeled. Hybridization probes enable detection of DNA or RNA sequences that are complementary to the probe sequence. These type of probes are now often referred to as “TaqMan probe, Molecular Beacons, Scorpions, or LightCycler probes.

    Linear hydrolysis probes consist of oligonucleotides with a fluorescent label on the 5’-end and a quencher molecule on the 3’-end.

    (1)   Probes are designed to have an annealing temperature above that of the primers.

    (2)   As the reaction is cooled from the melting temperature above that of the primers,
            the probe hybridizes to the target sequence.

    (3)   Cooling further, the primer hybridizes and the new strand is elongated until the DNA
            polymerase reaches the 5’ end of the probe.

    (4)   The probe is cleaved by 5’-3’ exonuclease activity of the enzyme and the fluorescent
            label is released.

    (5)   Theoretically, a fluorescent label should be released with each amplicon synthesized.

    However, in a real experiment, between 4 to 47 % of amplicons are detected when using theses probes. Different probe methods are known to have different sensitivities of detection due to different efficiencies in separating the fluorescent label from the quencher. It has been observed that the insertion of a bridged nucleic acid (BNA/LNA) into a linear hydrolysis probe can increase the detection sensitivity by up to 10-fold.


    Scorpion Probes


    Scorpion probes offer a different detection system. Scorpion probes combine a forward primer and the detection probe into a single molecule. The fluorophore and quencher are held in close proximity with a stem structure. First, the primer region hybridizes and elongates from the single-stranded target. The Scorpion opens when the template is melted away and the probe region hybridizes to the target region. This hybridization reaction separates the label from the quencher.

     

    BNA based PCR Probes


    The composition and sequence context of the selected target sequence for a PCR probe influences the design of the probe. Molecular probes used for diagnostic assay development need to be designed accordingly to allow for the assay to be of high sensitivity and specificity. Certain fundamental rules need to be adhered to when designing an accurate quantitative PCR assay. The composition of primers and probes dictated how well an assay works. For example, the melting temperature of the primer and probe duplex is determined by their sequence and length. The addition of a fluorophore and spacers can also have an influence on the melting temperature. If the nature of the desired target sequence does not support a design according to guidelines the development of a diagnostic assay may be impaired.


    BNA-containing oligonucleotides useful for RT-Q-PCR assays can range in length between 12 and 20 nucleotides (
    nt). This is significantly shorter than unmodified primers and probes displaying the same Tm. Furthermore, primers and probes modified with BNA monomers provide greater flexibility in designing consensus primers and probes for the detection of partially homologous target sequences, such as related viral species and serotypes.

    Finally, BNA-modified oligonucleotides in RT-Q-PCR assays reveal a specificity and sensitivity superior to other types of primers and probes.

     

    General guidelines for BNA oligonucleotide design

     

    (1)   Add BNA monomers at the site where specificity and discrimination are required.
            For example, in allele-specific probes at the SNP position or at the end of allele-
            specific primers.

    (2)   For blocking or clamping probes place one BNA monomer per every 3 bases within
            an oligomer. For example in a 20mer oligomer, about 4 or more 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 were
            found to be necessary to recruit RNase H. For diagnostic application, the modification
            of one nucleotide (
    nt) with a BNA nucleotide may be enough.

    (3)   The spacing of the BNA monomers need not be different within the oligonucleotide
            sequence used for different applications. For example, if the spacing is appropriate
            the same BNA monomer may be used.

    (4)   The use of no more than 4-8 BNA’s within a 20mer probe is recommended but is
            depended on the specific application
    .

    (5)   Long BNA stretches can have high affinity and interactions between nucleotides
            can cause the oligonucleotide to fold onto itself. This can produce a secondary
            structure, therefore using
    runs of three or more G bases are generally not
            recommended.

    (6)   Avoid BNA:BNA interactions that cause self-complementarity or are complementarity
            to other BNA-containing oligonucleotides.

    (7)   Each BNA–NC monomer increases the Tm by about 4 to 5 degrees Celsius.
            Using this information allows estimation of the Tm of the BNA containing
            oligonucleotide.

    (8)   BNA-NC(NMe) modifications add 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.

    (9)   Blocks of BNA near the 3’ end of primers can prevent polymerase activity.

    (10) Keep the GC content between 30 to 60%.

     

    General design guidelines for Real-Time qPCR probes

     

    (1)   The Tm of dual-labeled probes is typically slightly higher than the primer annealing
            temperature, approximately 65 to 70 °C.

    (2)   The optimal length of BNA modified dual-labeled probes is 14 to 18 nucleotides.
            Even shorter more efficient probes may be obtained by careful design.

    (3)   Maintain Tm values for BNA labeled probes to match the Tm of the corresponding
            longer DNA probes.

    (4)   Start with substituting every third base with BNAs in the central segment of the probe.
            Between 4 to 6 BNA substitutes may be required to obtain a useful Tm.

    (5)   Avoid stretches of more than 3 G DNA or BNA bases.

    (6)   To enable detection of single-nucleotide mutations (SNPs) select the probe sequence
            so that the mutation is located centrally in the probe. Start with a single BNA at the
            SNP location, a triplet covering the mutation, or make a short BNA probe.
            Do not position the SNP at the very ends of the probe.

    (7)   Always check for possible secondary structures in the probe and avoid BNA/BNA
            interactions.

    (8)   Position the dual-labeled probe as close as possible to the forward primer.

    (9)   Avoid G in the 5’-position next to the fluorophore. Guanosine (G) can quench adjacent
            fluorophores.

    (10)  Select the strand with the lowest amounts of Gs in the probe.

    (11)  Avoid longer stretches of identical nucleotides.

    (12)  Keep the GC content between 30 to 60%.

    (13)  To allow for exonuclease cleavage always add one DNA in the 5’-end of the probe.

     

    BNAs in PCR Primers

     

    (1)   BNAs in PCR probes can increase the binding affinity and specificity of PCR primers.
            For example, at the 3’ end in allele-specific PCR and in the SNP positions in allele-
            specific hybridization probes.

    (2)   However, not all working DNA primer pairs may be enhanced with the addition of BNAs.

    (3)   BNAs should be considered as a supplemental tool for the design of primer pairs.

    (4)   Adding BNAs to the primer will increase Tm leading to an increased affinity which allows
            the shortening of the primer sequence.

    (5)   BNAs may be used to adjust the Tm of primers to match PCR-cycling conditions.

    (6)   BNAs should be added to the 5’ prime region where modifications result in an increase
            of binding affinity but will not negatively affect specificity primarily determined by the
            3’ end.

    (7)   BNA spiked primers may allow for the discrimination of highly homologous, alternatively
            spliced isoforms.

    (8)   For allele-specific primers add only one BNA at the 3’end.

    (9)   Avoid stretches of more than four (4) BNAs.

    (10)  A typical 18mer may not contain more than 3 or 4 BNAs.

    (11)  Do not use blocks of BNA at the 3’ end.

    (12)  Keep the GC content between 30 to 60%.

    (13)  Avoid stretches of more than 3 G DNA or BNA base.

    (14)  The Tm of the primer pairs should be nearly equal.

    (15)  Typical Tms of PCR primers for dual-labeled assays are in the range from 58 to 60°C.

    (16)  For allele-specific primers, a single BNA should be placed in the terminal 3’ or the
             3’-1 position.

     

    BNAs in Blocker Probes – BNA Clamping

     

    (1)  Add 3’ phosphate or amino group or, better, any other spacer group, such as a
           (-C3-OH spacer), to prevent extension by the polymerase.

    (2)  For detection blocker or clamping probes, there are no exact design rules. Adjust
           the Tm by adding BNA bases to allow the correct allele probe or the primer to
           bind with the desired affinity.

    (3)  For primer-blocking probes position the discrimination site in the 3’ region.
           Add BNAs in this region.

    (4)  For PCR blockers design the probe to allow only the PCR blocker probe binding
           to avoid general PCR inhibition.

      

    Other special applications for BNAs

     

    • MicroRNA (miRNA) detection.
    • Universal Probe Libraries.
    • Special diagnostic PCR.
    • Affinity Capture Probes.

     

    Glossary 

    Tm

    Melting temperature: The melting temperature is the temperature in °C at which 50% of the oligonucleotide and its perfect complement are in a duplex.

    Td

    Dissociation temperature: The dissociation temperature is the temperature in °C at which 50% of an oligonucleotide and its perfect filter-bound complement are in duplex at the particular salt concentration and total strand concentration.

    ΔH 

    Enthalpy: The Enthalpy change can be calculated by subtracting the bond energies of the product from the bond energies of the reactions.

    ΔS 

    Entropy: The change in entropy is the tendency for randomness in a system. Natural systems have the tendency toward low enthalpy and high entropy.

     

    Reference

     

    Sreenivas Chavali, Anubha Mahajan, Rubina Tabassum, Souvik Maiti and Dwaipayan Bharadwaj;  Oligonucleotide properties determination and primer designing: a critical examination of predictions.Bioinformatics. Vol. 21 no. 20 2005, pages 3918-3925. doi:10.1093/bioinformatics/bti633

     

    Sung-Kun Kim, Klaus D. Linse, Parker Retes, Patrick Castro, Miguel Castro; Bridged Nucleic Acids (BNAs) as Molecular Tools. Journal of Biochemistry and Molecular Biology Research 2015; 1(3): 67-71
    Available from: URL:  
    http://www.ghrnet.org/index.php/jbmbr/article/view/1235/1527

     

    Nolan and Bustin: PCR Technology, Current Innovations. 3rd edition. CRC Press. 2013.

     

    Alejandro Panjkovich and Francisco Melo; Structural bioinformatics - Comparison of different melting temperature calculation methods for short DNA sequences. Bioinformatics. Vol. 21 no. 6 2005, pages 711–722.

     

    SantaLucia, J., Jr, Allawi, H.T., Seneviratne, P.A. 1996; Improved nearest-neighbor parameters for predicting DNA duplex stability. Biochemistry353555–3562.

     



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    The promising clinical results with the human monoclonal antibodies aducanumab and solanezumab targeting
    β-amyloid in Alzheimer’s disease treatment, confirm both the amyloid cascade hypothesis and protective natural
    immunity, while strengthening the immunotherapeutic approach. That aducanumab recognizes a conformational
    epitope formed by oligomers emphasizes the need for whole β-amyloid, not just its B-cell epitopes as have been
    the norm to avoid pro-inflammatory Th1-reactions.That truncated β-amyloid having N-terminal pyroglutamate is
    present only in diseased brain implies a new useful vaccine antigen. Another relevant antigen is the tau protein,
    which shows a close association and cooperativity with β-amyloid in exacerbating this disease. Hence, effective
    vaccines may be polyvalent, presenting to the immune system a number of antigens relevant to induce an immune
    response to prevent or slowdown the onset of this disease. The presence of both B and T cell epitopes in the antigens,
    require a sole Th2 immunity to avert brain inflammation; a task that cannot be attain with adjuvants that under
    any conditions induce Th1 and/or Th17 immunities. Hence, new vaccine adjuvants are need to safely induce Th2
    while inhibiting Th1 immunity, an objective that can be achieved with certain fucosylated glycans or triterpene
    glycosides, which apparently bind to the DC-SIGN lectin on dendritic cells polarizing the immune response toward
    Th2 immunity. Because the triterpene glycosides have the pharmacophore needed to co-stimulate T cells, they may
    ameliorate the T-cell anergy associated with immunosenescence and responsible for poor vaccine efficacy in the
    elderly population, a critical issue for an Alzheimer’s vaccine.
     
     
    Open PDF to read the full article
     

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    What are MicroRNAs or miRNAs?


    miRNAs are a class of endogenous small non-coding RNAs (ncRNAs) approximately 21 to 24 nucleotides in length found in plants and animals including humans. MiRNAs have recently emerged as key regulators in many biological pathways. MiRNAs function in the post-transcriptional regulation of gene expression. Processing of miRNAs occurs from approximately 70 nucleotides in size hairpin precursor RNAs by the protein Dicer. miRNA have been shown to regulate their target messengerRNA (mRNA) by destabilizing mRNA molecules and translational repression. More than half of all messenger RNAs (mRNAs) are estimated to be targets of miRNAs. Each miRNA molecule is predicted to regulate up to hundreds of targets. miRNA appear to regulate a broad range of cellular processes, including proliferation, differentiation, and apoptosis by interacting with specific mRNAs through complementary base-pairing.




    Figure 1: The miRNA biogenesis pathway. MiRNAs are transcribed by RNA polymerase II as precursor RNAs from intergenic, intronic or polycistronic genomic loci in combination with specific transcription factors (TF). Following transcription, the transcripts are processed in the nucleus by the RNase III enzyme Drosha in complex with DGCR8 (DiGeorge syndrome chromosomal region 8) into pre-miRNAs. Pre-miRNAs are exported into the cytoplasm by the protein Exportin 5. Pre-miRNAs are further processed by the RNAs III enzyme Dicer together with TRBP (TAR RNA binding protein encoded by the TRBP gene) into a duplex consisting of a guide strand (miRNA) and passenger strand (miRNA*). Next, the mature miRNA is loaded into the RNA-induced silencing complex, or RISC, and acts as a guide strand that based on sequence complementarities recognize target mRNA. RISC inhibits translation or promotes destabilization of target mRNAs.

    Large scale production of miRNAs can be achieved by in-vitro transcription (IVT) with the help of T7 RNA polymerase and fully complementary ds DNA oligonucleotides containing a T7 promoter.

    The expression of miRNAs is post-transcriptionally regulated by modulation of their maturation. Initially miRNAs are transcribed with much longer RNAs called precursor microRNAs (pri-miRNAs). These are subsequently processed in a series of steps to produce the mature miRNA. First, the long hairpin structure that contains the mature miRNA is cleaved from the primary transcript. The Microprocessor multiprotein complex enables this processing step. The protein complex distinguishes hairpins that contain miRNAs from other hairpin structures in the transcriptome. The excised hairpin is exported to the cytoplasm. In the cytoplasm, the nuclease Dicer cleaves the mature miRNA from the precursor hairpin (pre-miRNA). The pre-element structure is removed. This structure contains a short stem-loop, the terminal loop and a short region of predicted base pairing, also known as the terminal loop region or apical region. The remaining duplex associates with an Argonaute protein. The single-stranded miRNA is then retained in the active miRNA-Argonaute complex.

    An understanding of factors that regulate miRNA expression is important for the development of therapeutics that target disease-related miRNAs. To achieve this, Chiravil et al. have solved part of the structure for miR-21 pre-element. Figure 1 shows an NMR based model of a miRNA pre-element, miR-21 pre-element. miR-21 is a miRNA that is elevated in both cancer and heart disease. Furthermore, it is highly expressed in a variety of tumors, contributing to the cancer phenotype by lowering translation of tumor suppressor genes. miR-21 is also expressed in hypertrophic heart tissue, where it contributes to the fibrotic response to cardiac stress or injury (Sara Chirayil, Qiong Wu, Carlos Amezcua, Kevin J. Luebke; NMR Characterization of an Oligonucleotide Model of the MiR-21 Pre-Element. Plos One 2014, 9, 9, e108231, page 1 to 11).


    Figure 2: Oligonucleotide model of miR-21 pre-element. Chirayil et al. in 2014 reported the NMR model of an oligonucleotide stem-loop structure based on the pre-element of an oncogenic miRNA, miR-21. Only part of the molecule is shown in the structural model since the secondary structure of the native sequence could only be poorly defined by NMR. The replacement of two putative G•U base pairs with G•C base pairs allowed the researchers to observe the imino proton resonance in the molecule and to solve the structure of the stem-loop. The predicted stem-loop structure is cleaved from the precursor of miR-21 (pre-miR-21) by the nuclease Dicer. This structure is also recognized by the protein complex that converts the primary transcript (pri-miR-21) into the pre-miRNA.

    Selected references to review:

    miRNAs - http://blog-biosyn.com/2013/08/30/are-micrornas-the-new-players-in-metabolism-and-metabolic-disorders/

    DGCG8  - http://www.ncbi.nlm.nih.gov/gene?Db=gene&Cmd=ShowDetailView&TermToSearch=54487;

    TRBP     - http://www.phosphosite.org/proteinAction.do?id=15651&showAllSites=true).

    BSI blog:  

    http://blog-biosyn.com/2013/08/30/are-micrornas-the-new-players-in-metabolism-and-metabolic-disorders/


    Matthieu P. M. H. Benoit, Lionel Imbert, Andrés Palencia, Julien Pérard, Christine Ebel, Jérôme Boisbouvier and Michael J. Plevin; The RNA-binding region of human TRBP interacts with microRNA precursors through two independent domains. Nucl. Acids Res. (2013), 1-12. doi: 10.1093/nar/gkt086. http://nar.oxfordjournals.org/content/early/2013/02/21/nar.gkt086.full.

    Bredy TW,Lin Q,Wei W,Baker-Andresen D,Mattick JS.; MicroRNA regulation of neural plasticity and memory. Neurobiol Learn Mem. 2011 Jul;96(1):89-94. doi: 10.1016/j.nlm.2011.04.004. Epub 2011 Apr 18.

    Sara Chirayil, Qiong Wu, Carlos Amezcua, Kevin J. Luebke; NMR Characterization of an Oligonucleotide Model of the MiR-21 Pre-Element.
    Plos
    One 2014, 9, 9, e108231, page 1 to 11.  http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0108231

    Gurtan AM,Sharp PA; The role of miRNAs in regulating gene expression networks. J Mol Biol. 2013 Oct 9;425(19):3582-600. doi: 10.1016/j.jmb.2013.03.007. Epub 2013 Mar 13.


    Amy E. Pasquinelli; MicroRNAs and their targets: recognition, regulation and an emerging reciprocal relationship. Nature Reviews Genetics13, 271-282(April 2012) | doi:10.1038/nrg3162. http://www.ncbi.nlm.nih.gov/pubmed/22411466

    Wilson RC, Tambe A, Kidwell MA, Noland CL, Schneider CP, Doudna JA; Dicer-trbp complex formation ensures accurate mammalian microrna biogenesis. . Mol Cell. (2015) 57 p.397.

     

     

     


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