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Latest Articles of Bio-Synthesis Inc.

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    Even though fluorescence in situ hybridization - FISH probe has been widely used in cytogenetic applications, it is generally time-consuming and lack of sensitivity and resolution. Bio-Synthesis's high affinity BNA enchanced FISH Probes (BFISH) provides an alternative solution for a fast, sensitive and specific detection of chromsomal DNA.

    You design. We can Help

    We are happy in assist you with BNA FISH probes. Please contact us for more information. YOu may also design your own BNA FISH probes using guideline below.
     

    •  Detection probes are typically 20-25 nucleotides in length. However, shorter or longer probes can also be used.
    • Avoid stretches of 3 or more Gs or Cs. Avoid stretches of more than 4 BNA bases, except when very short (9-10 nt) oligonucleotides are designed.
    • Avoid BNA with self-complementarity since BNA hybridizes very tightly to other BNA residues.
    • Keep the GC-content between 30-60 %.
    • A Tm of approximately 75 °C is recommended.
    • No BNA bases should be placed in palindromes (G-C base pairs are more critical than A-T base pairs).
    Other useful links:
    1. Can you suggest a BNA experimental condition for FISH application?
    2. Do you have suggested buffers for BNA experiment?
    3. Bridged Nculeic acid Design Guidelines
    4. BNA for Duplex and Triplex Formation
    5. Allele Specific BNA Primer Design

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  • 08/07/13--00:00: Oligo Biotin Labeling
  • Biotinylated Oligonucleotides

    Bio-Synthesis offers biotinylated oligonucleotides for RNA and DNA detection or isolation application due to its high affinity toward strepatvidin. Biotinylated oligonucleotides can be used to attach specifically to streptavidin-enzyme-conjugates, to streptavidin-protein-conjugates, to strepavidin coated surfaces or to streptavidine-dye-conjugates. Applications using biotinylated oligonucleotides include:
    • Immuno assay
    • Chemiluminescence assays
    • Fluorescence in-situ hybridisation FISH
    • Immunohistochemistry
    • Straptavidin affinity chromatography
    • Magnetic bead capture for cDNA screening
    Biotin (or vitamin H) is a small biologically active molecule with a molecular weight of 244,31 Da. It acts as a co-enzyme in living cells. Biotin can be coupled to oligonucleotides at the terminal and internal position at multiple sites.  Iin 2000, Sabnayagam et al have shown that the size of the DNA or RNA oligonucleotide covalently attached to biotin seems to limit the binding capacity to immobilized streptavidin and the ability to capture large DNA fragments with magnetic streptavidin beads was directly correlate to the arm length of the biotin. Therefore, we also offer long chain biotin to increase binding capacity of large DNA fragment, improve binding kinetics and the accessibility to enzymatic events  at the solid phase surfaces1.

    One drawback of using biotinylated oligonucleotide in the detection system is the likelihood to obtain high backgrounds due to the presence of natural biotin in living cells. Alternate detection methods are the use of  digoxigenin/antidigoxigenin assays or enzyme/substrate assays that require labelling of the oligonucleotide with Digoxigenin or HRP (Horseradish-Peroxidase). Bio-Synthesis also offers dignoxigenin oligonucleotide labeling and HRP-oligonucleotide conjugation services.

    Reference
    1. Sabanayagam et al., Nucleic Acids Res., 2000, 28, e33

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    Post-translational modifications of proteins play critical roles in the regulation and function of many known biological processes. Proteins can be post-translationally modified in many different ways, and a common post-transcriptional modification of lysine involves methylation. Lysine can be methylated once, twice, or three times by lysine methyltransferases. The transfer of methyl groups from S-adenosyl methionine to histones is catalyzed by enzymes known as histone methyltransferases. Histones which are methylated on certain residues can act epigenetically to repress or activate gene expression.

    Histones can undergo many modifications. One of these modifications, lysine methylation, has important functions in many biological processes that include transcriptional regulation, heterochromatin formation, and even X-chromosome inactivation.

    Histone lysine methylation occurs on histones H3 and H4, and can signal either transcriptional activation or repression, depending on the sites of methylation. Methylation on the same site can also lead to different outcomes depending on the number of methyl groups added to lysine’s side chain.

    The methylation affects the binding of surrounding DNA to those histones, because the effective radius of the positive charge is increased, reducing electrostatic attraction with the negatively charged DNA. Also, methyl groups themselves are hydrophobic and will alter the structure of water in the vicinity.

    Though widely associated with histones and gene expression, lysine methylation has also found other uses such as creating protein crystallizations crucial in X-ray structure determination.

    Crystallography is used widely to solve atomic detail of structures of macromolecules. However, success is dependent on obtaining diffraction-quality crystals. The hydrophobic nature of methylated lysines will favor protein-protein interactions and drive formation of certain protein complexes. The change in chemistry of the free amide group also affects other biophysical properties, like reducing the solubility of proteins and sometimes changing the oligomeric state. The effects of lysine methylation on behavior of proteins in crystallization can be complex; but can significantly improve success rate.

    With Lysines methylation being one of the most common post-translational modifications, BSI has dedicated great amount of resources to developing and offering all of the methylated lysine modifications. BSI is able to produce peptides with mono-methylated Lysine, Di-methylated Lysine, and tri-methylated Lysines. All modified peptides have to pass stringent QC and QA procedures and include MALDI-TOF, analytical HPLC, and C of A documentation.

    Contact us for Methylated Peptide Synthesis

    Reference:

    1. Martin, Zhang. The Diverse Functions of Histone Lysine Methylation. Molecular Cell Biology, Volume 6, 838- Nov 2005 Nature Reviews
    2. Walter, et al. Lysine Methylation as a Routine Ways & Means Rescue Strategy for Protein Crystallization Structure 14, 1617–1622, November 2006 Elsevier 849

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    Oligonucleotide Analyzer

    The quantity of an oligonucleotide usually is given as its total optical density (OD value), as amount of substance (nmol) or as its available mass (μg). The OD value is measured experimentally, then the other values are calculated.

    OD Value

    The OD value of a sample at a wavelength of 260 nm is defined as the extinction that occurs in measuring absorption of the sample in 1 ml aqueous solution in a 1 cm cuvette at the respective wavelength.

    In practice, OD values are often outside of the linear measurement range of an UV spectrometer; therefore diluted samples have to be quantified and to be extrapolated to the total OD value.

    Example:

    The dried oligonucleotide is dissolved in 400 μl of sterile water. From this stock solution an aliquot of 10 μl is taken and filled up with sterile water to 1 ml. Now the extinction value of this diluted solution is measured in a photometer in a 1 cm quartz cuvette at a wavelength of 260 nm. The obtained extinction (e.g. 0,25) is multiplied with the aliquotation factor (400 μl / 10 μl = 40). The result is the OD value (10 in our example).

    Amount of oligonucleotide in nmol

    According to the law of Lambert and Beer (E = Epsilon * C * d) one can convert from the extinction E (OD value) to the concentration C and therefore to the amount of substance of the oligonucleotide. Strictly speaking, the extinction coefficient Epsilon is different for each oligonucleotide sequence and would have to be ascertained empirically. Using so-called nearest-neighbour methods the extinction coefficient of an oligo can also be obtained computationally with acceptable precision. In a good approximation it correspondents to the sum of the extinction coefficients of the individual nucleotides in the sequence.Using the oligo's sequence data and the OD value one can calculate the amount of material as

    follows:

    n [ nmol ] = 100
     -----------------------------------------------------------
    1,54 X A + 1,17 X G +n [OD] X C + 0,92 X T

    n [OD]: OD value
    n [nmol]: Amount in nmol
    A,G,C,T: Number of the respective bases in the oligonucleotide

    For mixed sequences the following values for the amount of material (nmol) result depending on oligo length:

    OD Value Oligo length (Number of nucleotides
    10 20 30 40 50 60 70 80 90 100
    1 9,4 4,7 3,1 2,3 1,9 1,6 1,3 1,2 1,0 0,9
    3 28 14 9 7 5,6 4,7 4,0 3,5 3,1 2,8
    10 94 47 31 23 19 16 13 12 10 9
    25 234 117 78 59 47 39 33 29 26 23
    100 937 468 312 234 187 156 134 117 104 94

    Thus, for a rough estimate applies for a mixed 20mer oligonucleotide :

    1 OD approximates 5 nmol (5000 pmol).

    Amount of oligonucleotide in μg

    Using the amount of substance in nmol and the oligo's molecular weight, one can calculate the amount of oligonucleotide in μg:

    n [ μg ] =   n [ nmol ] X MW [ g / mol ]
      ----------------------------------
    1000

    n [μg]: Amount in μg
    n [nmol]: Amount in nmol
    MW: Molecular weight

    Also, for rough estimates applies:

    1 OD approximates 30 μg DNA oligonucleotide

    Calculating concentrations

    1. Concentration adjustment to x pmol/μl

    A dried oligonucleotide is to be dissolved to obtain a concentation of x pmol/μl.

    v [ μl ] =  1000 X n [ nmol ]
      ----------------------------------
    c [ pmol / μl ]

    n [nmol]: Amount in nmol
    c [pmol/μl]: Concentration
    v [μl]: Volume of the oligo

    Example:

    An oligonucleotide (54 nmol) is to be dissolved with a target concentration of 50 pmol/μl in the resulting solution: v[μl] = 1000 * 54 nmol / 50 pmol/μl = 1080 μl

    Remark:

    The following concentrations are identical:

    c[ pmol / μl]= c[nmol / ml]= c[μmol / l]= c[μM ]
    Example:

    100 pmol/μl = 100 nmol/ml = 100 μmol/l = 100 μM

    2. Concentration when dissolving in a defined volume:

    A dried oligonucleotide is to be dissolved in a defined volume of water. Which concentration results ?

    c [ pmol / μl ] =  1000 X n [ nmol ]
      ----------------------------------
    v [ μl ]

    n [nmol]: Amount of substance in nmol
    c [pmol/μl]: Concentration
    v [μl]: Volumen of the oligos

    Example:

    An oligonucleotide (54 nmol) is to be dissolved in 200 μl water. Which concentation will result ? c[pmol/μl] = 1000 * 54 nmol / 200 μl = 270 pmol/μl

    Calculation of the molecular weight

    The molecular weight of an oligonucleotide is calculated from the number of individual nucleotides in the oligonucleotide and from possible modifications of the oligonucleotide:

    MWoligo=313,2*A + 329,2*G + 289,2*C + 304,2*T + MWmod -61 * [g/mol]

    A,G,C,T: Number of bases in the oligo

    MWmod: Molecular weight of a modification, if present

    Example:

    5’-CCA GGC AGT CTT ATT TTG ACT-3’

    MW = 313,2 *4 +329,2 *4 + 289,2 * 5 + 304,2 * 8 –61 = 6388,2 g/mol

    Melting temperature Tm

    The melting temperature Tm of a DNA double strand is defined as the temperature at which 50% of the double strand have been denaturated, thus being present in single stranded form. Length of the oligo, base composition and concentration of an oligonucleotide as well as the salt concentration in the solution have a key influences on the Tm value. There are different more or less exact methods of predicting the melting temperature:

    a) Wallace Rule (2 + 4 Rule) [1]

    This rule which is valid for very short oligonucleotides (up to about 15 bases) assumes a contribution of 2 °C for each AT-pair and of 4°C for each GC-pair to the melting temperature of a double strand:

    Tm = 2°C ×(A + T )+ 4°C ×(G + C)

    The rule was established for hybridizations to membrane-bound oligonucleotides and assumes a salt concentration of 1 M. For solution-based experiments one should add 8°C to the calculated temperature.

    b) Calculation by GC-content

    The following formula based on Howly et al. [2][3] takes mostly into account the GC content of the oligo and is valid for long oligonucleotides:

    Tm = 81,5 + 0,41 (%GC) + 16,6 log c(M+) – 500/n –0,61 (%F) –1,2 D

    with

    %GC = Percentage of GC-pairs
    c(M+) = Concentration of mono-valent cations
    n = Number of nucleotides
    %F = Percentage of formamide in the buffer
    D = Percentage of mismatches

    c) Nearest-Neighbor Method

    The so-called Nearest-Neighbor-Method also takes into account sequence-dependent stacking effects for Tm calculation and is based on thermodynamic properties of neighbouring nucleotide pairs. This method is reliable for oligonucleotides of medium length (20 – 60 bases).

    Formula [4]:

    Tm = [(1000 x dH) /(A + dS + R x ln (C/4))] – 273.15 + 16,6 x log c(K+)

    with

    Nearest-Neighbor MethoddH = Sum of the enthalpies of all pairs
    dS = Sum of the entropies of all pairs
    A = -10.8 cal, Entropy of helix formation
    R = 1.984 cal/grad x mol, Gas constant
    C = Oligonucleotide concentration (250 pmol/l)
    c(K+) = Concentration of potassic ions in the oligo solution (50 mmol/l)

    Thermodynamic data [5][6]:
    Dinucleotide Enthalpy dH (kcal) Entropy dS (cal)
    AA -9.1 24.0
    AG -7.8 -20.8
    AC -6.5 -17.3
    AT -8.6 -23.9
     
    GA -5.6 -13.5
    GG -11.0 -26.6
    GC -11.1 -26.7
    GT -6.5 -17.3
     
    CA -5.8 -12.9
    CG -11.9 -27.8
    CC -11.0 -26.6
    CT -7.8 -20.8
     
    TA -5.8 -16.9
    TG -5.8 -12.9
    TC -5.6 -13.5
    TT -9.1 -24.0
    Example:

    Oligo: 5'-GTC GAA CCG GAA ACC ACC CCT-3'

    dH = -6.5 –5.6 –11.9 –5.6 –9.1 –6.5 –11.0 –11.9 –11.0 –5.6 –9.1 –9.1 – 6.5 –11.0 -5.8 -6.5 –11.0 –11.0 –11.0 –7.8 = -173.5 dS = -17.3 –13.5 –27.8 –13.5 –24.0 –17.3 –26.6 –27.8 –26.6 –13.5 –24.0 –24.0 –17.3 –26.6 –12.9 –17.3 -26.6 –26.6 –26.6 – 20.8 = -430.6

    Tm = (1000 x (–173.5))/ ((-10.8 –430.6 + 1.984 x ln (6.25 x 10-11)) – 273.15 + 16.6 log 50x10-3 = 60.7

    All methods for the calculation of melting temperatures given above do not take into account modifications of the oligonucleotide (e.g. dyes, linkers).

    Literature
    1. R.B. Wallace, J. Schaffer, R.F. Murphy, J. Bonner, K.Itakura; Nuc. Acids Res. 1979, 6, 3543
    2. P.M. Howley, M.F. Israel, M-F. Law, M.A. Martin, M.A. J. Biological Chemistry 1979, 254, 4876
    3. R. Teoule, H.Bazin, B.Fouqué, A.Roget, S.Sauvaigo; Nucleosides & Nucleotides 1991, 10, 129
    4. W. Rychlik, W.J. Spencer, R.E. Rhoads; Nuc. Acids Res. 1990, 18, 6409-6413
    5. K.J. Breslauer, R. Frank, H. Blöcker, L.A. Marky; Proc. Natl. Acad. Sci 1986, 83, 3736-3750
    6. P.N. Borer, B. Dengler, I.J. Tinoco, O.C. Uhlenbeck; J. Mol.Biol. 1983, 86, 843-853
    7. 10/13


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    Template optimization using In Vitro Transcription

    Bio-Synthesis provides enzymatic long RNA synthesis using transcription from DNA template. In vitro transcribed RNA can be used as control template for Taqman SNP genotyping assays, drug metabolism genotyping assays or as positive control template for basic clinical research.

    The development of an organism depends upon accurate and finely-tuned control of mRNA transcription. The round worm Caenorhabditis elegans (C. elegans) is a transparent nematode about 1 mm in length. This organism has been studied intensively in recent decades and has become a model organism for molecular biologists. Already various biological processes that involve direct regulation or specific functions of general transcription machinery components such as “Mediator”, or transcription co-factors that function in multiple discrete regulator systems, such as chromatin modification proteins, have been identified. For example, the multiprotein complex termed Mediator functions as a transcriptional coactivator in all eukaryotes. This protein complex was discovered by Roger D. Kornberg who was awarded the 2006 Nobel Prize in Chemistry for “his studies of the molecular basis of eukaryotic transcription". This multiprotein complex sometimes is also referred to as the Vitamin D Receptor Interacting Protein (DRIP) coactivator complex or the Thyroid Hormone Receptor-associated Proteins (TRAP). However, all genes have to be expressed in order to function in a cell. The first step in expression is transcription of the gene into a complementary RNA strand. For genes that code for transfer RNA (tRNA) and ribosomal RNA (rRNA) moleculs the transcript itself is the functionally important molecule. For other genes however, the transcript is translated into a protein.

    The Central Dogma in molecular biology for prokaryotic in comparison to eukaryotic cells is outlined as follows: In prokaryotic cells, which have no nuclear membrane, DNA replication and transcription and RNA translation occurs in one compartment. Furthermore, the three processes can occur simultaneously. However, in eukaryotic cells, which have a nuclear membrane, DNA replication and transcription occurs in the nucleus, and proteins are synthesized in the cytoplasm. RNA molecules therefore have to travel across the nuclear membrane before translation can happen. In these cells transcription and translation are physically separated. In addition, the primary transcript, heterogeneous nuclear RNA (hnRNA), is post-transcriptional processed to generate a messenger RNA (mRNA) molecule that can migrate through the nuclear membrane. The following figure illustrates this.

    transcription, IVT, and template optimization

    Advancements made in recent years in manual and automated oligonucleotide synthesis technologies allow now to routinely synthesize DNA, RNA and/or artificial oligonucleotides that can be modified or unmodified with high purity. Artificial oligonucleotides can act as RNA and DNA mimics but may have different sets of properties, such as higher affinities to their targets. These technologies have already enabled the use of synthetic oligonucleotides to construct synthetic biochemical circuits from simple components useful for the study of in-vitro transcriptional circuits. For example the use of two essential enzymes, bacteriophage T7 RNA polymerase and Escherichia coli ribonuclease H, together with synthetic oligonucleotides allowed the systematic construction of arbitrary circuits for the study of synthetic in-vitro transciption. In addition, scientists in recent years have shown that mathematical modeling can be used to guide the design process of this type of systems. Some of these experimental conditions yielded oscillating biochemical circuits. The notion here is that synthetic transcriptional oscillators could prove valuable for systematic exploration of biochemical circuit design principles. The understanding of these principles could allow scientist to design artificial cells and control nanoscale devices. Future studies will surely identify additional examples of direct communications between regulatory and general transcription factors and reveal how promoter groups and gene networks are regulated through these interactions. Finally, new insights into the specific biological processes in which they are involved will be gained.

    What exactly is transcription?

    Transcription refers to the transfer of genetic code information from one kind of nucleic acid to another. It refers to the process by which a base sequence of messenger RNA is synthesized by an RNA polymerase on a template of complementary DNA. On the other hand, the reversal of this process or flow of information is called “reverse transcription.” In reverse transcription the normal pattern is reversed, for example by a viral enzyme called “reverse transcriptase.” Furthermore, a polymerase associated with the process of transcription is called a “transcriptase”. The DNA-dependent RNA polymerase is an example for a transcriptase. The template is defined as “a single-stranded polynucleotide or the region of a polynucleotide that directs the synthesis of a complementary polynucleotide”. Experiments that aim to find out which portion of a DNA molecule is transcribed into RNA are called “transcript analysis”. Furthermore, the entire mRNA content of a cell or tissue is now called the “transcriptome”.

    In bacteria, or prokaryotes, transcription is catalyzed by a single RNA polymerase. In e.coli the RNA polymerase is a large enzyme of almost 500 kilodaltons. The holoenzyme has five subunits, α2ββ’ω, and the core enzyme lacks the σ factor. An essential step in bacterial transcription is the binding of one of a number of dissociable accessory proteins, called the σ factor, to a core RNA polymerase to form a holoenzyme. In e. coli the core enzyme, α2ββ’ω with a mass of 379 kDa binds the σ70 factor to form the holoenzyme α2ββ’ω σ70, with a mass of 449 kDa. Only as a holoenzyme can RNA polymerase initiate transcription and it is thought that the holoenzyme binds weakly to the DNA and explores the DNA double helix until with the help of the σ factor it recognizes a promoter sequence to which it finely binds tightly. Finn et al. in 2000 determined the structures of the core RNA polymerase and the σ70 holoenzyme using cryo-electron microscopy and angular reconstruction.

    Escherichia coli RNA polymerase (RNAP) is the most studied bacterial RNA polymerase and has been used as the model RNAP for screening and evaluating potential RNAP-targeting antibiotics. Murakami in 2013 reported the X-ray structure of the E. coli RNAP σ(70) holoenzyme which showed the σ region 1.1 (σ1.1) and the α subunit C-terminal domain in the context of an intact RNAP. The structure revealed that σ1.1 is positioned at the RNAP DNA-binding channel and completely blocks DNA entry to the RNAP active site. Furthermore, the σ1.1 contains a basic patch on its surface. It is thought that this batch may play an important role in DNA interaction to facilitate open promoter complex formation. In this structure the α-subunit C-terminal domain is positioned next to σ domain 4 with a fully stretched linker between the N- and C-terminal domains. The X-ray based model is depicted in the next figure.

    RNA polymerases

    Transcription involves RNA polymerases binding, initiation,elongation and termination. During the initiation of the transcription cycle an RNA polymerase searches for a promoter site and binds to DNA, unwinds and separates the two strands. The separation occurs in such a way that one strand, the template strand, is copied, but not the other. Next, a base pair forms between a base in the template strand and a ribonucleotide triphosphate. The first nucleotide retains its three phosphate groups at the 5’ end as the RNA chain grows on the OH group attached to its 3’ carbon. During elongation the RNA strand grows from its 5’-end to its 3’ end, as the polymerase copies the template DNA strand from its 3’-end to its 5’-end. The RNA polymerase catalyzes the formation of a phosphodiester bond. The DNA duplex unwinds and a newly duplex is formed made of the newly synthesized RNA and DNA template strand, which extends for 10 to 12 bases behind the most recent DNA. Next, the growing RNA molecule detaches from the template and the DNA helix forms again. The new RNA strand has exactly the same sequence as the nontemplate strand of DNA, however, wherever DNA has a thymidine (T), the RNA has a uridine (U). During termination, the ending of chain growth, the RNA polymerase detaches from the DNA template and the growing RNA chain. Termination sites or sequences in the DNA signal the end of transcription. Many prokaryotic genes contain self-complementary sequences that can fold back on itself and form hairpin duplexes or stretches of U’s.

    However, termination in eukaryotes is more complex and the elucidation of the exact mechanism or mechanisms is still under investigation. Termination can also occur at termination sites that do not have these features, in additon, for this to occur protein factors are needed. One of these termination assisting proteins is called rho (ρ). In addition, so called antiterminating proteins can allow elongation to continue through DNA sequences that otherwise can serve as termination sites.

    Transcription of two genes

    Transcription of two genes. RNA polymerase moves from the 3’ end of the template strand and creates an RNA strand that grows in a 5’ to 3’ direction because it must be antiparallel to the template strand. Some genes are transcribed from one strand of the DNA double helix whereas other genes are transcribed from the other as the template. Furthermore, a uracil is being added to the 3’ end of the transcript for gene 1. Querying the Pubmed structure data base revealed that many solved structures are available for polymerase complexes indicating the importance of these protein complexes.

    Zhang et al. in 2012 have determined crystal structures at 2.9 and 3.0 Å resolution of functional transcription initiation complexes comprising Thermus thermophilus RNA polymerase, sigma factor A (σ), and a promoter DNA fragment corresponding to the transcription bubble and downstream double-stranded DNA of the RNAP-promoter open complex. The structures showed that σ recognizes the –10 element and discriminator element through interactions that include the unstacking and insertion into pockets of three DNA bases and that RNA polymerase recognizes the –4/+2 region through interactions that include the unstacking and insertion into a pocket of the +2 base. Furthermore the structures also revealed that interactions between σ and template-strand single-stranded DNA (ssDNA) preorganize the template-strand ssDNA to engage the active center of RNA polymerase. The model of the crystal structure of the bacterial RNA polymerase (RNAP)-promoter complex (RPo) in complex with a ribonucleotide primer (RPo-GpA) is shown below.

    promoter complex

    Model of the bacterial RNA polymerase (RNAP)-promoter complex (RPo) in complex with a ribonucleotide primer (RPo-GpA).

    The crystal structure of a bacterial RNA polymerase (RNAP)-promoter complex (RPo) in complex with a ribonucleotide primer (RPo-GpA) is illustrated at the left part of the panel. The interactions of RNAP and the σ factor with the transcription-bubble non-template strand, transcription-bubble template strand, and the downstream dsDNA is shown at the right part of the panel. (Source: Zhang et al. 2012). The sigma factor (σ factor) is a protein needed for the initiation of RNA synthesis. This bacterial transcription initiation factor enables specific binding of RNA polymerase to gene promoters. However, each specific sigma factor used to initiate transcription of a given gene will vary depending on the gene and on the environmental signals needed to initiate transcription of that gene. In addition, every molecule of RNA polymerase holoenzyme contains exactly one sigma factor subunit.

    Bye 1 is a transcription factor that links histones to post-translational modification events.

    Kinkelin et al. in 2013 reported crystal structures of the nuclear protein bypass of Ess1 (Bye1) TFIIS-like domain (TLD) bound to Pol II and three different polymerase II-nucleic acid complexes. The researchers could show that like TFIIS, Bye1 binds with its TLD to the polymerase II jaw and funnel. Furthermore, it was demonstrated that Bye1 is recruited in vivo to chromatin via its TLD and occupies the 5'-region of active genes. The paper showed that a plant homeo domain (PHD) in Bye1 binds histone H3 tails with trimethylated lysine 4. This interaction is enhanced by the presence of neighboring posttranslational modifications (PTMs) that mark active transcription and is impaired by repressive PTMs. The scientists identified putative human homologs of Bye1, the proteins PHD finger protein 3 and death-inducer obliterator. Both proteins are implicated in cancer. These results establish Bye1 as a chromatin transcription factor that links histones with active PTMs to transcribing Polymerase II. Bypass of Ess1 (Bye1) is a nuclear protein with a domain resembling the central domain in the transcription elongation factor TFIIS.

    What allowed the scientists to establish their model?

    The researchers used purified cloned proteins together with Surface Plasmon Resonance, crystallization of the complexes followed by determining the X-ray structure, chromatin fractionation, histone peptide microarrays, synthetic lethality screening, in vitro transcription assays, RNA extension assays, chromatin immune precipitation (ChIP) and gene averaged profiling. A model of this complex is shown next.

    polymerase II-Bye 1-nucleosome complex
    Model of a polymerase II-Bye 1-nucleosome complex.

    This model shows that Bye associates with active genes in front of the +2 nucleosome. The model is based on crystal structures and ChIP occupancy peak positions (Source: Kinkelin et al., 2013).

    The central dogma of molecular biology first stated by Francis Crick in 1958 and re-stated in a Nature paper published in 1970 describes the “information flow in biological systems”. It deals with the detailed residue-by-residue transfer of sequential information. It states that such information cannot be transferred back from protein to either protein or nucleic acid and has been also described as "DNA makes RNA makes protein." Furthermore, since it is a simplification it does not make it clear that the sequence hypothesis as stated by Crick does not preclude the reverse flow of information from RNA to DNA, but only the reverse flow from protein to RNA or DNA.

    central dogma

    This graphics (left) illustrates the central dogma and its expansion as new knowledge about the nature of different types of RNAs became available.


    The flow of information and substances in a cell is even more complex. This is illustrated in more detail next. Regulatory feedback loops are depicted as well.

    exponential increase

    The exponential increase in RNA research has in recent years tremendously increased our knowledge about their function. This has led to our expanded understanding of complex regulatory pathways implied by the central dogma. The next graphic shows the current view how RNA processing is thought to occur in a cell.

    Eukaryotes express

    Eukaryotes express many functional non-protein-coding RNAs (ncRNAs) that used to be thought of as “junk DNA’ or the “dark matter” of the DNA. These RNAs participate in the processing and regulation of other RNA molecules common patterns have emerged that form a network-like RNA infrastructure. For more details see Collins & Penny in Trend in Genetics, 2009.

    How can we study RNA transcripts of genes?

    Over the years scientists have devised a variety of techniques to study RNA transcripts. Some of them detect the presence of a transcript and give some information of its length whereas others enable the start and end of the transcript to be mapped and the position of introns to be located. The following is a list of these methods:

    1. Northern hybridization: RNA molecules present in RNA extracts are separated by electrophoresis, for example, in an agarose gel, using denaturing buffers to ensure that the RNAs do not form inter- or intramolecular base pairs. After electrophoresis, the gel is blotted onto a nylon, a nitrocellulose or a polyvinylidene fluoride (PVDF) membrane followed by hybridization with a labeled probe. If the probe is a cloned gene, the band that appears on the color or radioactively developed membrane is the transcript of the gene. The size can be determined from its position within the gel in relation to RNA marker molecules or RNA isolated from different tissues that are run in different lanes of the gel. This allows to find out if the gene is differentially expressed.
    2. Transcript mapping by hybridization between gene and RNA: DNA-mRNA hybridization can be used to investigate if incomplete or complete cDNA synthesis occurred. If a hybrid is formed between a DNA strand that contains a gene and its mRNA the boundaries between double- and single-stranded regions will mark the start and end points of the mRNA. Introns that are present in the DNA but not in the mRNA will “loop out” as additional single-stranded regions. S1 nuclease degrades single-stranded DNA or RNA polynucleotides including the single-stranded ends of predominantly double-stranded molecules. However S1 nuclease has no effect on double-stranded DNA or on DNA-RNA hybrids. Singel-stranded DNA fragments protected from S1 nuclease digestion can be recovered by treatment with alkali. S1 nuclease mapping allows the localization of the starting-point and end-point of a transcript.
    3. Transcript analysis by primer extension: Primer extension can only be used if at least part of the sequence of a transcript is known. In this technique a short oligonucleotide primer must be annealed to the RNA at a known position. Usually the primer anneals within 100-200 nucleotides of the 5’ end of the transcript and is extended by reverse transcriptase which is a cDNA reaction. The 3’ end of the newly synthesized strand of DNA corresponds with the 5’ terminal end of the transcript. Determination of the length of the single-stranded DNA molecule and correlating this information with the annealing position of the primers allows location of the position of this terminus.
    4. Transcript analysis by PCR: A modified method of a standard reverse trancriptase PCR procedure called rapid amplification of cDNA ends (RACE) can be used to identify the 5’ and 3’ termini of RNA molecules and allow to map the ends of a transcript. Several RACE methods have been developed over the years.
    5. Digital PCR can be used to determine the number of transcripts: Farago et al. in 2003 have shown that digital PCR can be used to determine the number of transcripts from single neurons after patch-clamp recoding.

    How can the yields of full-length RNA from an in-vitro transcription reaction be maximized?

    The quality and quantity of RNA produced in an in-vitro transcription reaction is dependent upon several factors. The size of the RNA transcript, template concentration, reaction time and temperature, all influence the yield of the final full-length transcript. During an individual application, the starting template and the resulting transcript determine how a reaction needs to be modified to increase the final yields. In general, T7, T3, or SP6 RNA polymerase are used for in-vitro transcription reactions. Each polymerase recognizes a short, well-defined, phage promoter sequence with a high degree of specificity that has a minimal length of 20 nucleotide bases. In-vitro transcription reactions usually include ribonucleoside tri-phosphates (rNTPs) at concentrations of 0.5 mM per each nucleotide, reaction buffer, a linear DNA template, amounts of 1 to 2 µg, and the appropriate bacteriophage RNA polymerase. On average, these reactions produce 10 to 40 µg of RNA. However, since increasingly techniques such as gene expression profiling using microarrays, RNA interference (RNAi) gene-silencing, in-vitro translation, ribozymes, and RNA structure studies require larger quantities of RNA commercial in-vitro transcription kits have been developed to address this need. Starting with 1 µg of template up to 100 or 200 µg of RNA may be produced. Even though reaction conditions in commercial kits have been optimized to maximize the RNA yield from control templates which typically can produce 1 to 2 kB transcripts, each unique transcript may need adjustments to the protocol to maximize yields.

    Template preparation: The DNA template used should be linear, double-stranded, and relatively clean. Typically templates used include linearized plasmids that contain blunt or 5’-protruding ends, PCR products or cDNAs. The templates should be free of RNase and other contaminations such as phenol, trace metals and sodium dodecyl sulfate (SDS). The treatment of the DNA with proteinase K, followed by phenol-chloroform extraction and ethanol precipitation usually allows producing a sufficiently clean template. The analysis of the template with the help of an agarose or polyacrylamide gel and staining with ethidium bromide or syber green allows verifying that the template is pure enough.

    Maximizing yield for long RNA transcripts:

    Template concentration and reaction time: For the production of a long-transcript a higher concentration of template DNA is less critical. Sometimes increasing the amount of template reduces the total reaction time to yield the RNA transcript.

    Optimizing long transcripts from limited amounts of template: If the amounts of template DNA is limited to less than 1 µg the reaction time and/or temperature may need to be increased, or the reaction may need to be scaled up, to maximize yields. Reaction incubation times may need to be increased to 2 to 3 hours.

    Reaction temperature for a long transcript: Increasing the reaction temperature from 37 °C to 42 °C will increase the rate at which the RNA is produced and also increases the maximal yield that can be attained. This temperature effect is more significant for lower template concentrations. A limited amount of template may demand that the reaction is incubated at 42 °C for 3 to 4 hours.

    Scale-up for long transcripts: To achieve even higher yields all components of the reaction mixture including the template may need to be scaled up by 6 to 10 fold. Because the initiation of the reaction is the rate-limiting step in a transcription reaction, the reaction dynamics in a short-transcript reaction differ from those in long-transcript reaction. The best way to optimize transcription of short RNAs is to maximize the number of possible transcription initiations. This can be achieved by increasing template concentration or reaction times.

    Template size and concentration for a short transcript: The increase of the template concentration in an in-vitro transcription reaction will result in an increase of the yield of a short RNA transcript. Usually in-vitro transcription reactions use 1 µg of template. However, if a 75 base pair (bp) double-stranded oligonucleotide template is used 20 picomoles of DNA will be used. On the other hand, if 1 µg of a 4.2 kilo base (kb) linear plasmid is used only 360 femtomoles of DNA is present. Therefore, 1 µg of an oligonucleotide template produces more short RNA than 1 µg of a plasmid template if all other parameters are equal in the reaction.

    Reaction time for a short transcript: Increasing the reaction time will increase the number of transcription initiation events and significantly increase the yield of the RNA. However, after a reaction time of 3 to 4 hours the yield in general will plateau.

    Reaction temperature for a short transcript: Typically in-vitro transcription reactions are performed at 37o°C. The increase of the temperature to 42 °C can increase the yields and shorten reaction times.

    To conclude, RNA yields for both long and short transcripts from in-vitro transcription reactions can be maximized by adjusting the template DNA concentration, the reaction time, and the reaction temperature.

    Template preparation:

    Transcription templates can either be synthetic DNA, PCR products, or linearized vectors. Synthetic templates can be purified using reversed phase HPLC, ion exchange chromatography, an oligonucleotide purification cartridge, desalted by butanol precipitation or ethanol precipitation, or purified by Urea-PAGE. Furthermore, synthetic templates can be used for the antisense strand (cDNA).

    References

    Brown, T.A.; Gene cloning and DNA analysis. An introduction. Sixth edition. Wiley-Blackwell. 2010.

    Crick, F.H.C. (1958): On Protein Synthesis. Symp. Soc. Exp. Biol. XII, 139-163.

    Crick, F (August 1970). "Central dogma of molecular biology". Nature 227 (5258): 561–3. Bibcode:1970 Nature.227..561C. doi:10.1038/227561a0. PMID 4913914.}

    Collins LJ, Penny D.;The RNA infrastructure: dark matter of the eukaryotic cell? Trends Genet. 2009 Mar;25(3):120-8. doi: 10.1016/j.tig.2008.12.003. Epub 2009 Jan 24.

    Nóra Faragó, Ágnes K. Kocsis, Sándor Lovas, Gábor Molnár, Eszter Boldog, Márton Rózsa, Viktor Szemenyei, Enikő Vámos, Lajos I. Nagy, Gábor Tamás, and László G. Puskás; Digital PCR to determine the number of transcripts from single neurons after patch-clamp recording. BioTechniques 54:327-336 ( June 2013) doi 10.2144/000114029.

    Robert D. Finn, Elena V. Orlova, Brent Gowen, Martin Buck, and Marin van Heel; Escherichia coli RNA polymerase core and holoenzyme structures. The EMBO Journal vol. 19 No. 24 pp. 6833-6844, 2000.

    Magali Frugier, Catherine Florentz, Mir Wais Hosseini, Jean-Marie Lehn and Richard Giegd; Synthetic polyamines stimulate in-vitro transcription by T7 RNA polymerase2784-2790 Nucleic Acids Research, 1994, Vol. 22, No. 14.

    Kinkelin, K., Wozniak, G.G., Rothbart, S.B., Lidschreiber, M., Strahl, B.D., Cramer, P.; Structures of RNA polymerase II complexes with Bye1, a chromatin-binding PHF3/DIDO homologue. (2013) Proc.Natl.Acad.Sci.USA 110: 15277.

    Jongmin Kim and Erik Winfree; Synthetic in-vitro transcriptional oscillators. Molecular Systems Biology 7:465

    Lewin’s Genes I to XI. Oxford University Press.

    Lodish, Harvey; Molecular Cell Biology – 2nd to 7th edition. Scientific American Books.

    Craig T. Martin, Daniel K. Muller, and Joseph E. Coleman; Processivity in Early Stages of Transcription by T7 RNA Polymerase. Biochemistry 1988, 27, 3966-3974.

    Murakami KS; X-ray crystal structure of escherichia coli RNA polymerase sigma70 holoenzyme. J.Biol.Chem. (2013) 288 p.9126.

    Patrushev LI, Bocharova TN, Khesin RB.;The effect of various templates and oligonucleotide primers on RNA and poly (A) synthesis by E. coli and T7 RNA polymerases. FEBS Lett. 1978 Feb 1;86(1):108-12.

    Yu Zhang, Yu Feng, Sujoy Chatterjee, Steve Tuske, Mary X. Ho, Eddy Arnold, Richard H. Ebright; Structural Basis of Transcription Initiation. Science 338, 1076 (2012).

    10/28/2013


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    EXTINCTION COEFFICIENTS AND FLUORESCENCE DATA

    Calculate extinction coefficient of an oligo by either summing up the extinction coefficients of the individual bases times their number of occurrences. Or use a formula that takes into account nearest neighbor effects. An algorithm for this calculation can be found on the web. Just type in the sequence and the program will calculate the concentration of a l A260/ml solution.

    To calculate the MW of the aminomodified oligo just add 179.16 to the calculated MW of the unmodified oligo.

    CAT. NO. Nucleoside l max-1 Emax-1 l max-2 Emax-2 E260
        (nm) (ml/µmole) (nm) (ml/µmole) (ml/µmole)

    10-1001

    7-deaza-dA

    270

    11.3

     

     

    9.4

    10-1003

    N6-Me-dA

    266

    16.9

     

     

    15.2

    10-1006

    Etheno-dA

    295

    3.4

    274

    5.9

    4.7

    10-1007

    8-Br-dA

    266

    16.4

     

     

    14.8

    10-1008

    8-Oxo-dA

    268

    12.2

     

     

    11.1

    10-1014

    pdC

    295

    7.7

    234

    14.7

    5.1

    10-1017

    Pyrrolo-dC

    339

    2.36

    229

    17.5

    2.41

    10-1021

    7-deaza-dG

    259

    12.6

     

     

    12.6

    10-1027

    8-Br-dG

    253

    12.1

     

     

    11.3

    10-1028

    8-oxo-dG

    294

    5.2

    250

    6.7

    5.9

    10-1031

    5'-OMe-dT

    266

    9

     

     

    8.3

    10-1035

    Carboxy-dT

    297

    16.1

    261

    14.7

    14.7

    10-1036

    2-thio-dT

    278

    17.5

    220

    14.8

    10

    10-1040

    dI (Inosine)

    249

    12.5

     

     

    7.5

    10-1041

    dNebularine

    262

    7.1

     

     

    7.0

    10-1043

    3-Nitropyrrole

    283

    8.8

     

     

    7.7

    10-1044

    5-Nitroindole

    328

    8.5

    265

    17.0

    16.0

    10-1045

    4-Methylindole

    265

    7.9

     

     

    7.2

    10-1046

    2-Aminopurine

    303

    6.8

    243

    5.7

    1.0

    10-1047

    dP

    294

    6.7

    231

    7.4

    2.9

    10-1048

    dK

    279

    10.7

     

     

    7.7

    10-1050

    dU

    262

    10.0

     

     

    10.0

    10-1052

    4-thio-dU

    330

    30.4

     

     

    3.6

    10-1053

    5-OH-dU

    280

    7.8

     

     

    4.9

    10-1054

    pdU

    291

    11.3

    231

    11.4

    3.5

    10-1055

    d-pseudoU

    262

    7.7

     

     

    7.6

    10-1060

    5-Me-dC

    277

    9.0

     

     

    5.7

    10-1061

    5-Me-dZ

    314

    4.8

    218

    8.6

    1.8

    10-1063

    5-OH-dC

    292

    6.3

    220

    13.3

    3.4

    10-1065

    5-Me-isodC

    260

    6.3

     

     

    6.3

    10-1067

    5-Me-isodC

    260

    6.3

     

     

    6.3

    10-1076

    7-deaza-dX

    284

    6.5

    252

    10.4

    8.8

    10-1077

    iso-dG

    292

    11.0

     

     

    4.6

    10-1078

    iso-dG

    292

    11.0

     

     

    4.6

    10-1080

    5-Br-dC

    287

    6.0

     

     

    3.1

    10-1081

    5-I-dC

    293

    5.7

     

     

    3.3

    10-1085

    2,6-diaminoPurine

    278

    10.2

    255

    9.3

    8.5

    10-1090

    5-Br-dU

    278

    9.7

     

     

    5.1

    10-1091

    5-I-dU

    287

    7.7

     

     

    3.7

    10-1094

    Furano-dT

    See plot

    10-1095

    2,4-difluoro-toluene

    266

    2.3

     

     

    1.8

    10-1097*

    AP-dC

    362

    10.5

     

     

    10.9

    10-1530

    dihydro-dT

    210

    6.3

     

     

    <0.1

    10-1550

    dihydro-dU

    210

    6.3

     

     

    <0.1

    10-1550

    dihydro-dU

    210

    6.3

     

     

    <0.1

    Note: Biotin and Cholesterol have no absorbance at 260nm.

    *With an extinction coefficient of approximately 10,500 M-1 and a quantum yield of fluorescence of 0.2, AP-dC is 2-3 times as bright as our popular Pyrrolo-dC analog. In addition, AP-dC exhibits a Stokes’ shift greater than 100 nm. As with most fluorescent base analogs, it is substantially quenched upon forming a duplex. The quantum yield drops to 0.1 while gaining significant structure in the emission spectrum (Figure 4), making it an ideal probe of DNA structure.

    FLUORESCENCE DATA

    Dye E 260 nm E lambda max Excitation max Emission max QY Notes
      (L/mol cm) (L/mol cm) (nm) (nm)    
    Acridine 39,500 9,120 421 497    
    2-aminopurine 1,000 3,600 303 371    
    Cy3 4,930 136,000 547 563 0.15  
    Cy3.5 24,000 116,000 591 604 0.15  
    Cy5 10,000 250,000 646 662 0.3  
    Cy5.5 21,500 209,000 688 707 0.3  
    Dabcyl-dT 29,100 32,000 476      
    5'-Dabcyl 11,100 32,000 468      
    Eclipse Quencher 6,600 33,300 530 N/A 0  
    Etheno-dA 4,800 5,800 276 405 0.035  
    6-FAM 20,900 75,000 495 521 0.9  
    3'-(6-Fluorescein) 13,700   494 522    
    Fluorescein-dT 38,800 75,000 494 522 0.9  
    HEX 31,580 96,000 537 556 0.7  
    NBD 3,700 19,500 485 535 0.1  
    Psoralen 16,500 11,000 301      
    Pyrrolo-dC 4,000 3,700 345 470 0.07/0.02 QY 0.07 single-stranded; 0.02 ds, deprotected in ammonia 55°C ON
    Pyrene-dU 18,500 42,200 402      
    Redmond Red 12,100 74,000 (pH 9.1)
    52,300 (pH 7.1)
    579 595 0.84  
    TAMRA 32,300 89,000 556 580 0.7  
    TET 16,255 86,000 519 539 0.9  
    Yakima Yellow 23,700 83,800 530.5 549 0.96  

    PHYSICAL PROPERTIES OF BLACK HOLE QUENCHERS

    Quencher lmax (nm) E260 (L/mol.cm) Emax (L/mol.cm)
    BHQ-0 493 7,700 34,000
    BHQ-1 534 8,000 34,000
    BHQ-2 579 8,000 38,000
    BHQ-3 672 13,000 42,700

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    Pam2Cys-peptides and Pam3Cys-peptides are Palmitoylated Peptides known to activate the immune system through toll like receptors. Short peptide sequences play a major role in immune recognition. A pathogen, such as bacteria or a parasite, is captured when encountered and degraded by specialized antigen presenting cells of the immune system into short peptide sequences known as epitopes. Palmitoylated Peptides can be part of these peptide epitopes. These epitopes are then presented to effector T cells and, if the correct cellular signals are given, can trigger off a cascade of events that eventually induces an immune response. Synthetic peptides including Palmitoylated Peptides have been used for the design of vaccines in the past. If the epitope that is recognized by an antibody or a T cell is known, a vaccine can be designed around this epitope. Palmitoylated peptides that contain dipalmitoyl-S-glyceryl cysteine (Pam2Cys), tripalmitoyl-S-glyceryl cysteine (Pam3Cys) and macrophage-activating lipopeptide (MALP-2) moieties can be used to deliver antigenic peptides that contain toll like receptor (TLR) agonists. Palmitoylated peptides that contain Pam2Cys target the TLR1-2 receptor and palmitoylated peptides that contain Pam3Cys target the TLR2-6 receptor. TLR1 recognizes peptidoglycans and lipoproteins together with TLR2 as a heterodimer and is found on the surface of macrophages and neutrophils. Palmitoylation is a post translational modification in which fatty acids, such as palmitic acid, are attached to cysteine and sometimes to serine and threonine residues of proteins. This modification enhances the hydrophobicity of proteins and contributes to their membrane association. Furthermore, these types of modified peptides appear to be a promis­ing approach for the development of new immunotherapeu­tics. A chemically synthesized tripalmitoyl pentapeptide (TPP) has been shown to be a potent mitogen and polyclonal activator for B lymphocytes of both lipopolysaccharide (LPS)-R and LPS-LR mice. Immunogenicity of vaccine peptides is dependent on how the peptide antigen and adjuvant are recog­nized by the immune system. For the development of a specific pep­tide-based vaccine formulation that is efficiently delivered to its specific target factors such as peptide orientation and structure, stability of peptides against deg­radation and clearance, tissue localization, antigen uptake and processing, and toxic­ity have to be considered. The attachment of a single palmitic acid moiety to an antigenic peptide to form a palmitoylated peptide enhances peptide immunogenicity in a TLR2-dependent manner and the conjugation of antigenic peptides to dipalmitoyl-S-glyceryl cysteine (Pam2Cys) has generated antigen-specific immune responses in a wide variety of animal studies including those evaluating B‑cell epitopes for a contraceptive therapy, Tc‑cell epitopes for hepati­tis C virus, influenza virus, intracellular bacterium Listeria monocytogenes, and the model tumor antigen ovalbumin. Lipid-core peptides linking synthetic analogs of Pam3Cys to multiple peptides via dendrimeric mul­tiple antigen peptide structures have proven to be effective for Group A streptococcal (GAS) vaccines. In addition palmitic acid-based adjuvants using palmitic acid, dipalmitoyl-S-glyceryl-cysteine (Pam2Cys) and tripalmitoyl-S-glyceryl-cysteine (Pam3Cys) lipid moieties have been shown to be effective for epitope-based vaccines and do not exhibit the harmful side effects that are commonly associated with many other adjuvant formulations. This type of vaccine delivery system also allows for the delivery of multiple peptides in one complex. Furthermore, different epitopes that may be needed to achieve broad-spectrum immunity can be included. 

    10/13

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  • 10/29/13--00:00: RNA Transcription
  • Transcription refers to the transfer of genetic code information from one kind of nucleic acid to another and to the process by which a base sequence of messenger RNA is synthesized by an RNA polymerase on a template of complementary DNA. A polymerase associated with the process of transcription is called a "transcriptase". The DNA-dependent RNA polymerase is an example for a transcriptase. The template is defined as "a single-stranded polynucleotide or the region of a polynucleotide that directs the synthesis of a complementary polynucleotide". Experiments that aim to find out which portion of a DNA molecule is transcribed into RNA are called "transcript analysis", and the entire mRNA content of a cell or tissue is now called the "transcriptome". SP6, T7 and T3 phage RNA polymerases have high specificity for their respective base promoters. Due to the development of cloning vectors containing promoters for these polymerases the in vitro synthesis of single stranded RNA molecules is now a routine laboratory procedure. Sense or antisense RNAs can now also be synthesized either automatically using standard phosphoramidite chemistries or from sequences cloned into multiple cloning sites. Linearized plasmid sequences, PCR products and synthetic oligonucleotides can be used as templates for transcription reactions, the template need only be single stranded. In vitro transcription reactions are generally used for the synthesis of highly specific activity RNA probes and the synthesis of larger quantities of RNA. Large amounts of RNA are needed for in vitro translation, microinjection, ribozyme studies, microarray analysis, non-radioisotopic probes, the synthesis of high specific activity probes for use in ribonuclease protection assays, Northern and Southern blotting and in situ hybridizations, and a variety of other applications.

    10/29

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    Amino Acid Analysis / Testing refers to the quantitative analysis of either free amino acids or amino acids released from proteins via hydrolysis present in biological samples such as blood, plasma, urine, foods, dietary or health supplements, nutraceuticals, beverages, tablets, supplement capsules, plant extracts and any other samples or matrices that contain amino acids.

    Proteins, peptides and amino acids are found in a great variety of food sources including animals, fungi, vegetables, cereals and many others. It has been shown by many authors that due to their complexity the identification of these compounds require the use of advanced analytical techniques. Many techniques including high-performance liquid chromatography, sometimes referred to as high-pressure liquid chromatography (HPLC), gas chromatography (GC), capillary electrophoresis (CE), nuclear magnetic resonance (NMR), Fourier transform infrared spectroscopy (FTIR), inductively coupled plasma mass spectrometry (ICP-MS), inmunosensors, and others have been used or were investigated for their use to analyze amino acids in different matrices in the past. Furthermore, amino acids have been identified and quantified in different natural matrices using micellar electrokinetic chromatography (MEKC), micro chip electrophoresis or HPLC. The US Pharmacopeia Dietary Supplement Chapter lists detailed test methods for each individual amino acid ingredient as follows: the identification of amino acids is perform by FTIR, analysis of chromatographic impurities is performed by a TLC–ninhydrin based method and the determination of the potency of raw materials is done by using titration assays. However, these are classical or older methods. The newer, more recent methods that were developed during the last decades utilize automated instruments called “amino acid analyzers.” Most amino acid analysis or testing labs are now capable of routinely testing amino acids as either the free amino acids or those released from protein hydrolysates.

    Amino acid analysis enables the precise determination of protein quantities and provides detailed information regarding the relative amino acid composition of proteins and free amino acids present in physiological samples. The resulting amino acid composition is a characteristic profile for a protein. In some cases this profile allows to identify a protein via database searches. A typical amino acid analysis includes a hydrolysis step followed by separation, and detection and quantification of the released and labeled amino acids. Briefly, samples that contain proteins are subjected to acid hydrolysis and are broken down into individual amino acids. Next, the released amino acids are reacted with a derivatizing reagent, converted to stable chromophore modified amino acids and are separated and analyzed by HPLC.

    Amino acids present in finished goods such as tablets, capsules, powders, and in liquid syrups can all be analyzed using automated amino acid analysis. The analysis of single and/or multiple amino acid preparations can be performed and the results are documented according to the specific guidelines.

    What are amino acids?

    Amino acids are the "building blocks" of proteins and are found in every human tissue as well as all mammalian tissues. Amino acids play major roles in nearly every chemical process in the human body. Amino acids are vital molecules that affect both physical and mental functions in mammals and humans.

    Proteins are found in the brain, the nervous system, ligaments, tendons, bones, as well as in antibodies, and are vital for the regulation of enzymes and blood transport proteins. Only twenty amino acids of the naturally occurring amino acids are found in proteins because they are the only ones that are coded for by the genetic code.

    Chemically, an amino acid is an organic acid in which one of the hydrogen atoms on a carbon atom has been replaced by an ammonia group (NH2). The term amino acid usually refers to an aminocarboxylic acid. One particular type of amino acids called α-amino acids are of the general formula R-CHNH2-COOH with the NH2 group in the alpha position. All amino acids, except the amino acid glycine, are stereoisomers and only the L-form or L-enantiomer, which is one mirror image, of the possible two amino acids, is biologically active in an organism. The L refers to L-stereoisomers or left-handed isomers of the amino acids. These L-forms are the result of the hydrolysis products of proteins. All proteins consist of groups of different types and percentages of L-amino acids. Sometimes, in rare instances, these classes of molecules also include α–amino phosphoric acids and α–aminosulfonic acids. The following structures illustrate the nature of stereoisomers.

    Stereoisomers of alanine
    Stereoisomers of alanine

    The models of D- and L-alanine are used here as an example to describe the nature of stereo isomers. The four bonds of the central alpha carbon of an amino acid point towards the four corners of a tetrahedron. Alanine has a methyl group (CH3) as a side chain group. If we imagine holding the model of the structure with the carboxyl group (COOH) at the top and the amino group (NH2) at the bottom, the CH3 group in the D form will be on the right, and in the mirror image L form will be on the left.

    Amino acids are important for the function of an organism and are part of its metabolism. The misregulation of biochemical processes that involve amino acids in the human body can lead to metabolic diseases. An example is the presence of excessive amounts of specific amino acids in the blood called “aminoacidemia.” Another example is the excessive excretion of amino acids in the urine called“aminoaciduria.” In addition, an inherited disorder called hyperbasic aminoaciduria is associated with a deficiency of a dibasic amino acid transport. Individuals with this disorder do not display protein intolerance. Because of the great number of metabolic disorders that involve amino acids and the wide range of affected systems, nearly every "presented complaint" to a doctor may have a congenital metabolic disease as a possible cause. This is especially true in childhood. Therefore, it is safe to reason that amino acid analysis or testing is an important technique for the analysis of metabolites that contain primary and secondary amino groups in their structure, which are present in humans or mammals as well as in food stuff, nutraceuticals and health supplements. This type of analysis is also quite important in clinical chemistry.

    Except for the nine so called "essential amino acids," the human body can synthesize all of the amino acids necessary for the biochemical synthesis of proteins. Plants and micro-organisms can synthesize all amino acids from small precursor molecules. These nine amino acids must be included in the human diet or taken as supplements to be available in adequate amounts. The failure to obtain enough of even one of these essential amino acids can result in serious health implications and the degradation of the body's proteins. These phenomena can lead to the destruction of muscle and other protein structures to obtain the amino acids that the body needs to survive. Because of this, amino acids are classified into two main groups: essential and non-essential amino acids. There are nine essential amino acids in the group: phenylalanine, valine, threonine, tryptophan, isoleucine, methionine, leucine, lysine, and histidine. In some biochemical and molecular biology books, arginine and histidine are sometimes also listed as essential amino acids. Non-essential amino acids are amino acids that can be produced by the human body. These include: alanine, aspartic acid, arginine, cysteine, cystine, glutamine, glycine, serine, tyrosine, hydroxyproline, asparagine, and proline. However, non-essential amino acids are also used as a supplement for cell culture medium to increase cell growth and cell viability. Furthermore, protein sources that have the full complement of essential amino acids are sometimes called complete proteins. Whereas proteins that are devoid or deficient in one or more essential amino acid are called incomplete proteins. Arginine is essential during times of rapid growth. It is particularly needed during childhood or when muscle growth is initiated during weight training sessions. Furthermore, the amounts of each essential amino acid required depends on the overall amino acid composition of the proteins that are consumed. For example, human cells can synthesize cysteine from methionine if needed. However, if the intake of cysteine is low extra methionine is needed in the diet not only to meet the needed methionine levels but also to synthesize cysteine.

    For nutritional purposes, the protein quality can be estimated and a chemical score can be defined. Usually this is done by assigning a chemical score of one hundred (100) to whole egg protein and by comparing the proteins of interest to this protein. This score implies that the whole egg protein is completely utilized. The amino acid that gives the lowest score in relation to the reference protein will dictate the final chemical score. This amino acid is called the first limiting amino acid. The following formula is used to calculate the chemical score:

    Chemical score = [ amount of essential amino acid in test protein (mg/g)]
     ---------------------------------------------------------------------------------------------------------------------
    [ amount of same essential amino acid in reference protein or egg protein ]

    Proteinogenic amino acids are amino acids that are used by the cellular machinery of an organism to synthesize proteins. These amino acids are encoded for in the genetic code. There are 22 standard amino acids, but only 21 are found in eukaryotes. Selenocysteine and pyrrolysine are incorporated into proteins by different biosynthetic mechanisms. The other 20 are directly encoded by the universal genetic code.

    Branched-chain amino acids, a term used by the food and supplement industry, are amino acids that contain branched aliphatic side-chains. The side chains contain a carbon atom that is connected to more than two other carbon atoms. There are three branched-chain amino acids: leucine, isoleucine and valine. The structures of these amino acids are shown below.

    Leucine Isoleucine Valine
    Leucine Isoleucine Valine
    Branched-chain amino acids

    In muscle proteins these three branched-chain amino acids account for circa 35% of the essential amino acids and 40% of the preformed amino acids required by mammals.

    Amino acid profiles that determine the presence or absence and the quantities of the twenty amino acids in plasma can illuminate problems in amino acid status of a human patient. This is done by determining essential amino acid imbalances. Amino acid profiles containing up to 45 compounds can be even more informative. These evaluation types allow identifying the status of essential, branched chain, and other non-essential amino acids. Together, with the analysis of vitamins and minerals, this analysis allows identification of neuroendocrine, vascular, detoxification, and functional vitamin and mineral deficiencies.

    Fasting plasma amino acid levels represent a homeostatic balance between supply and utilization of amino acids, making amino acid analysis of plasma amino acids ideal for repeated assessments by monitoring the progress of treatment. Collecting a fasting plasma specimen from a patient removes recent dietary intake effects. The following factors can reflect changes over time in plasma: chronic dietary intake, digestive efficiency, hepatic uptake and skeletal muscle's ability to maintain transamination. Amino acid analysis or testing helps monitoring amino acid levels involved in nutritional function, including gastrointestinal function, cellular energy production, detoxification, neurotransmitter metabolism, muscle catabolism, collagen synthesis and maintenance, as nutritional markers, and can be indicators of vascular function. Furthermore, amino acid analysis or testing of amino acid levels in plasma is useful for the design of supplement formulas. For example, to manufacture customized amino acid blends based on a patient's specific test results. The notion here is that these customized amino acid formulations will provide appropriate amounts of essential and conditionally essential amino acids that can be delivered in a balanced ratio to offset the risk of imbalance, sometimes observed when single amino acid supplements are used. Known conditions associated with amino acid changes in plasma include cardiovascular disease, depression, anxiety, insomnia, chronic fatigue syndrome, multiple sclerosis, rheumatoid arthritis, epilepsy, congestive heart failure, impotence and erectile pain syndromes, multiple chemical sensitivities, detoxification disorders, autism spectrum disorders, Alzheimer's disease, hypothyroidism, arrhythmias, hypertension, ADD/ADHD, and infertility.   10/31/2013

    Table 1: The Genetically-Encoded Amino Acid Set
    1 Alanine, Ala, A 11 Methionine, Met, M
    2 Cysteine, Cys, C 12 Asparagine, Asn, N
    3 Aspartic acid, Asp, D 13 Proline, Pro, P
    4 Glutamic acid, Glu, E 11 Glutamine, Gln, Q
    5 Phenylalanine, Phe, F 15 Arginine, Arg, R
    6 Glycine, Gly, G 11 Serine, Ser, S
    7 Histidine, His, H 17 Threonine, Thr, T
    8 Isoleucine, Ile, I 18 Valine, Val, V
    9 Lysine, Lys, K 11 Tryptophan, Trp, W
    10 Leucine, Leu, L 20 Tyrosine, Tyr, Y
    Table 2: Other Common Amino Acids in Proteins
    1 Hydroxyproline (hydroxylated proline-two isomers)
    2 Cystine (oxidised cysteines)
    3 Pyroglutamic acid (cyclized N-terrninal glutamic acid)
    Table 3: Other Amino Acids Commonly used in Peptide Design
    1 Alpha-amino butyric acid (cysteine replacement)
    2 Beta-amino alanine (straight chain isomer of alanine)
    3 Norleucine (linear sidechain isomer of leucine)
    Table 3: Grouping by hydrophilicity and hydrophobicity
    Hydrophilic D, E, H, K, N, Q, R, S, T, hydroxyproline, pyroglutamic acid
    Hydrophobic A, F, I, L, M, P, V, W, Y, Alpha-amino butyric acid, beta-amino alanine, norleucine
    Indeterminate C,G
    Table 3: Other Groupings of Amino Acids
    Amino acids subject to oxidation under relatively mild conditions cysteine, methionine
    Amino acids subject to deamidation or dehydration asparagine, glutamine, C-terminal amides
    Amino acids subject to degradation during peptide preparation methionine, tryptophan
    Amino acids which can carry a positive charges lysine, arginine, N-terminal end of peptide, histidine
    Amino acids which can carry a negative charge aspartic acid, glutamic acid, C-terminal end of peptide, tyrosine

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    Thioate groups can be incorporate in the RNA linkages between two bases. This approach is dependent on the specific application.
    We recommend the incorporating of BNA monomers into synthetic oligonucleotides. BNA monomer contain a six member bridged structure with a unique N-O bond in the sugar moiety. This RNA analog was developed by Professor Emeritus Takeshi Imanishi of Osaka University. This moiety was designed to contain a nitrogen atom which is important in DNA chemistry as a conjugation site. It improves duplex and triplex stability by lowering the repulsion between negatively charged phosphate backbones.

    These nucleic acid analogs can be easily incorporated into natural oligonucleotide strands. They provide flexibility in designing BNA/DNA and BNA/RNA hybrid oligonucleotides to satisfy the need for very high and sequence-specific hybridization with natural nucleic acids. Additionally, they possess a strong nuclease-resistant property. While first generation BNA (also known as LNA) are still used in various applications, Bio-Synthesis Inc. now offers third generation, six membered bridged 2', 4' BNANCs which have shown to possess superior properties to the earlier generation of locked nucleic acids and peptide nucleic acid.

    BNA Advantages

    * Improved hybridization selectivity and specificity
    * Increased thermal duplex stability
    * Enhanced allelic discrimination
    * High biological stability to nuclease and protease
    * Superior antisense inhibition and potency
    * Flexible probe designs regardless of GC content
    * Easily adaptable to many DNA or RNA detection system 

    11/01/2013

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    Can jet lag be avoided? No, not yet, but recent research indicates that there is hope for a cure in the near future.

    The neuropeptide, arginine vasopressin, appears to be an important part of regulating our body clock. In a recent volume of the journal Science, Yamaguchi et al. showed that mice lacking receptors for the neuropeptide arginine vasopressin (AVP) are resistant to jet lag.

    Jet lag: Everybody who has traveled in airplanes around the globe or between continents through several time zones most likely experienced jet lag. Jet lag can disrupt the mental and physical well-being of the traveler. Therefore, jet lag highlights the importance of our internal "body clock." Unfortunately, so far there is no cure to avoid bodily symptoms caused by jet lag. Scientists now know that jet-lag symptoms arise from a temporal misalignment between the internal circadian clock and external solar time, and that our circadian system can sense and realign to a shifted cycle of light and darkness. New findings suggest that neuropeptide signals in the brain influence and participate in the regulation of the master circadian clock. This could be good news to help overcoming the health symptoms associated with jet lag.

    The circadian clock: Biological life, including the life of humans, is regulated by cyclic chemical processes in four dimensions. Humans perceive their surroundings usually as "spacetime", that is, we observe nature and the rooms we live in as three dimensions and the time we spend in them as a fourth dimension. Einstein went to great length to describe the nature of the three spatial dimensions and the impact of the single temporal dimension in his relativity theory. We all experience the cycle of day and night. Furthermore, almost all species exhibit daily changes in their behavior and/or physiology that are influenced by the earth turning on its axis and the daily rhythms of our solar system. However, these changes arise from a timekeeping system present within the organism. The timekeeping system, or biological "clock," enables an organism to anticipate and prepare for the changes in the physical environment that are associated with day and night cycles. This biological clock coordinates the internal temporal organization with the internal changes that take place in the organism. This ensures that the organism will "do the right thing" at the right time of the day. This endogenous circadian clock drives oscillations in physiology and behavior with a period of about 24 hours.

    The scientists in Yamaguchi research group found that circadian rhythms of behavior also called locomotor activity, clock gene expression, and body temperature immediately readjusted to phase-shifted light-dark cycles in mice lacking the vasopressin receptors V1a and V1b (V1a-/-V1b-/-). However, the behavior of V1a-/-V1b-/- mice was still coupled to the internal clock. It was found that the internal clock of the mice oscillated normally under standard conditions. The researcher's experiments with suprachiasmatic nucleus slices in culture suggested that interneuronal communication mediated by the vasopressin receptors V1a and V1b confers on the suprachiasmatic nucleus an intrinsic resistance to external perturbation. The pharmacological blockade of vasopressin receptors V1a and V1b in the suprachiasmatic nucleus of wild-type mice resulted in accelerated recovery from jet lag. This highlights the potential of vasopressin signaling as a therapeutic target for management of circadian rhythm misalignment, such as jet lag and shift work.
    The neuropeptid arginine vasopressin, also known as vasopressin, argipressin or antidiuretic hormone, is involved in a wide range of physiological regulatory processes, including water reabsorption, the regulation of blood pressure and water, glucose, and salt levels in the blood. Evidence suggests that it also plays an important role in social behavior, sexual motivation and bonding, and maternal responses to stress, as well as emotional status. The vasopressin hormone and the structurally related posterior pituitaty hormone oxytocin, are synthesized in magnocellular neurosecretory cells of the paraventricular nucleus and the supraoptic nucleus of the of the hypothalamus in most mammals. The peptide has the following amino acid sequence: Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Arg-Gly-NH2. Arginine vasopressin belongs to the family of neurohypophysial hormones. The peptide is derived from a preprohormone precursor molecule that is synthesized in the hypothalamus and stored in vesicles at the posterior pituitary from where it is released into the bloodstream. Before the peptides are released into the circulation on the basis of hormonal and synaptic signals, with assistance from the pituicytes, they are stored in Herring bodies.


    Reference

    Yoshiaki Yamaguchi, Toru Suzuki, Yasutaka Mizoro, Hiroshi Kori, Kazuki Okada, Yulin Chen,1 Jean-Michel Fustin, Fumiyoshi Yamazaki, Naoki Mizuguchi, Jing Zhang, Xin Dong, Gozoh Tsujimoto, Yasushi Okuno, Masao Doi, Hitoshi Okamura; Mice Genetically Deficient in Vasopressin V1a and V1b Receptors Are Resistant to Jet Lag. Science 342, 85 (2013).

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    Amino acid analysis of plasma metabolites.

    Amino acid analysis is an important tool for the analysis of free amino acids and other similar biomolecules that contain primary and secondary amino groups within their structure and which are present in blood serum, plasma, and other body fluids such as urine and cerebrospinal fluid (CSF). CSF is a clear, colorless bodily fluid found in the brain and spine. During the last decades, it has become clear that amino acids play a crucial role as metabolites and as regulators of metabolic pathways in mammals, including humans. Since the blood serves as a common medium that links all organs in the body together, plasma amino acid concentrations could be affected by metabolic disturbances in a particular organ system. Therefore, amino acid profiling of blood plasma samples can be used to study the levels of amino acid metabolites.

    Metabolite profiling usually refers to a set of metabolites, along with their concentrations, detected in a biological sample. Metabolite profiles can be very specific for certain classes of chemical compounds, such as lipids or amino acids. Similar to the genome, the full complement of genes of an organism, the metabolome, is described as the full complement of metabolites of an organism. The terms"metabolite" and"metabolome" have become established in the scientific literature since the year 2000. A Pubmed search for "metabolomics" showed that, as of Fall 2013, over 6,100 papers on this subject have already been published thus far. The combination of next-generation deep-sequencing technologies with mass spectrometry and other technologies such as amino acid analysis will surely provide more accurate data as a result of the extensive study of the metabolome in the near future.

    Abnormal profiles in amino acid concentrations are observed in various diseases such as liver disease, end-stage renal disease, hepatocellular carcinoma, and others. In addition, several plasma peptides have also been identified as hormones important in metabolic physiology and diseases. One important peptide class includes the family of cardiac natriuretic peptides. These types of peptides have emerged as potent metabolic hormones that exhibit a wide range of biological actions and are involved in the control of metabolic homeostasis. Homeostasis is the ability or tendency of an organism or cell to maintain internal equilibrium.  A mammalian body achieves this by adjusting all physiological processes to coordinate responses of its parts to any situation or stimulus that tends to disturb the normal conditions or functions in order to maintain internal stability. Therefore, changes in plasma amino acid levels can reflect the metabolic status of a patient. For example, patients with severe hepatic disease have an amino acid imbalance in which low levels of branched chain amino acids and high levels of aromatic amino acids are observed in their systemic blood when analyzed.  Further, it is known that the increase in aromatic amino acids levels in the brain can lead to a decrease in the normal neurotransmitters and an increase in the neurologically inactive phenylethanolamine and octopamine. The intake of branched chain amino acids improves the plasma amino acid balance. Peptides with high levels of branched chain amino acids and low levels of aromatic amino acids are called high-Fischer-ratio oligopeptides. The Fischer ratio is the ratio between branched-chain amino acids (BCAA) and aromatic amino acids (AAA) which can be defined by the formula Fr = (Leu + Val + Ile)/(Tyr + Phe). This ratio has been used for the diagnosis of hepatic encephalopathy and its drug treatment efficacy. High Fischer ratio oligopeptides can be derived from various food proteins and their resulting amino acid content can be determined by amino acid analysis.

    Reference

    Kimura T, Noguchi Y, Shikata N, Takahashi M Plasma amino acid analysis for diagnosis and amino acid-based metabolic networks. Curr Opin Clin Nutr Metab Care. 2009 Jan;12(1):49-53. doi: 10.1097/MCO.0b013e3283169242.

     
     

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  • 11/12/13--00:00: Conjugate Vaccines.

  • To elicit an immune response against protein/peptide antigens lacking T-cell epitopes, or against non-peptidic antigens, such as oligosaccharides, there is a need to conjugate these compounds covalently to a protein carrier that can supply T-cell epitopes in order to create a conjugate vaccine which triggers an effective immune response. Polysaccharides are not processed by “antigen presenting cells” or APCs, i.e. dendritic cells and macrophages, but they interact with B cells triggering antibody production in the absence of T cells, thus called T-independent antigens. Oligosaccharides are usually linked to a carrier protein to form conjugate vaccines, such as different inactivated bacterial toxins or toxoids, e.g. tetanus and diphtheria toxoids, to yield glycoconjugates, which are better immunogens than the oligosaccharides alone. Conjugation of the oligosaccharides to the carrier proteins would depend on their carbohydrate composition, for instance saccharides that carry carboxyl or amino groups can be conjugated to a protein using carbodimides. If the saccharide has only neutral sugars, it can be linked to a protein after a mild treatment with periodate to oxidize cis-hydroxyl groups and yield aldehyde groups, which can be linked to a protein’s amino group by reduction alkylation in the presence of cyanoborohydride. Alternatively, the saccharide can be linked to the protein by using either 1,1-carbonyldiimidazole (CDI) or 1-cyano-4-dimethyaminopyridinium (CDAP), to yield urethane and isourea bonds, respectively. However, while the conjugations with CDI need to perform in organic solvents that limit the saccharides’ solubility, conjugations with CDAP can be carried out in aqueous media, which allows good solubility for saccharides. As indicated before, peptides lacking T-cell epitopes can be linked covalently to proteins having those epitopes, such as serum albumin, edestin, thyroglobulin or KLH, using the carbodiimide method, alternatively, if the carrier protein has a carbohydrate it can be treated with periodate to oxidize the sugars and form an aldehyde, which can be linked by a primary amino group to the peptide. However, a potential problem of the periodate method is the formation of carrier protein aggregates, which may lead to insoluble aggregates.

    Another type of useful conjugate vaccine is that made by covalently linking a protein or peptide to an immune modulating agent or adjuvant; where the adjuvant moiety can be a CpG oligonucleotide, a lipid A derivative, a polysaccharide such as a mannan, or some other type of immune stimulatory agent that can be linked to the antigen. Depending on the nature and size of the antigen and the adjuvant it would be possible to use one of the conjugation methods available. Advantages of antigen-adjuvant conjugates are i) a more focused immune response, as the antigen and adjuvant would stimulate the same APC, thus requiring less amount of adjuvant and reducing the toxic effects associated with these compounds, ii) better specific-antigen antibody titers, and iii) depending on the antigen-adjuvant combination, they may overcome some genetic restrictions imposed on an antigen’s immune response. However, the benefits of using this type of conjugates would depend on the antigen and adjuvant used, which may limit the scope of this application.
      

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    This content provides several useful information of common biological constants and conversions. You can use these tables to aid your DNA/RNA/protein quantification, electrophoresis gel preparation, protein synthesis, calculating molecular mass and other research activities in molecular biology. Table include:

    Metric Prefixes


    Prefix Symbol Factor
    kilo k 103
    centi c 10-2
    milli m 10-3
    micro µ 10-6
    nano n 10-9
    pico p 10-12
    femto f 10-15
    atto a 10-18
    zepto z 10-21


    Spectrophotometric Conversion

    1 A260 unit of double-stranded DNA=50 µg/ml
    1 A260 unit of single-stranded DNA=33 µg/ml
    1 A260 unit of single-stranded RNA=40 µg/ml


    DNA Molar Conversions

    1 µg of 1,000 bp DNA 1.52pmol(3.03pmol of ends)
    1 µg of pBR322 DNA 0.36pmol DNA
    1 pmol of 1,000 bp DNA 0.66 µg
    1 pmol of pBR322 DNA 2.8 µg
    pBR322DNA:(4,361bp)

    Formulas for DNA Molar Conversions

    dsDNA
    To convert pmol to µg:
    pmol × N × 660 pg/pmol × 1 µg/106pg = µg
    To convert µg to pmol:
    µg × 106pg/1 µg × pmol/660pg × 1/N = pmol
    ssDNA
    To convert pmol to µg:
    pmol × N × 330 pg/pmol × 1µg/106pg = µg
    To convert µg to pmol:
    µg × 106pg/1 µg × pmol/330pg × 1/N = pmol
    where N is the number of nucleotides and 330 pg/pmol is the average MW of a nucleotide


    Protein Molar Conversions

    100 pmol of 100 kDa protein = 10 µg
    100 pmol of 50 kDa protein = 5 µg
    100 pmol of 10 kDa protein = 1 µg
    100 pmol of 1 kDa protein = 100 ng


    Protein and DNA Conversions

    1 kb DNA 333 amino acids
      37 kDa protein
    270 b DNA 10 kDa protein
    810 b DNA 30 kDa protein
    1.35 kb DNA 50 kDa protein
    2.7 kb DNA 100 kDa protein
    average MW of an amino acid 110 daltons
    Dalton (Da) is an alternate name for the atomic mass unit, and kilodalton (kDa) is 1,000 daltons. Thus a protein with a mass of 64 kDa has a molecular weight of 64,000 grams per mole


    Agarose Gel(%) of Linear DNA Resolution

    Recommended % Agarose Optimum Resolution for Linear DNA
    (Size of fragments in nucleotides;bp)
    0.5 1,000-30,000
    0.7 800-12,000
    1.0 500-10,000
    1.2 400-7,000
    1.5 200-3,000
    2.0 50-2,000


    Polyacrylamide Gel(%) of Potein Resolution

    Recommended % Acrylamide Protein Size Range
    8 40-200 kDa
    10 21-100 kDa
    12 10-40 kDa


    Length and M.W. of Common Nucleic Acids

    Nucleic Acid Number of Nucleotides Molecular Weight
    lambda DNA 48,502(dsDNA) 3.2 × 107
    pBR322DNA 4,361(dsDNA) 2.8 × 106
    28S rRNA 4,800 1.6 × 106
    23S rRNA(E.coli) 2,900 1.0 × 106
    18S rRNA 1,900 6.5 × 105
    16S rRNA(E.coli) 1,500 5.1 × 105
    5S rRNA(E.coli) 120 4.1 × 104
    tRNA(E.coli) 75 2.5 × 104
    *Molecular weights based on actual sequence.

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    Glutamic acid (Glu, E) is an amino acid with the formula HOOC-CH2-CH2-CH(NH2)-COOH and has a molecular weight of 147.13 g mol−1. The L-isomer is one of the basic building blocks of proteins. Glutamic acid is consumed in many prepared foods as a flavor additive in the form of monosodium glutamate. L-glutamic acid is a non-essential amino acid approved as a nutraceutical dietary supplement or micronutrient. The term “L-glutamate” describes any salt of L-glutamic acid. The sodium salt is called monosodium glutamate. Furthermore, human and animal studies indicate that glutamate is the major oxidative fuel for the gut where dietary glutamate is extensively metabolized by the intestinal enterocytes. In addition glutamate is an important precursor for bioactive molecules, including glutathione, and functions as a key neurotransmitter. When taken orally, L-glutamate is absorbed from the lumen of the small intestine into the enterocytes. The absorption of this amino acid is efficient and occurs by an active transport mechanism. Enterocytes are intestinal absorptive cells that are present in the small intestines, colon and appendix. The glycocalyx surface coat consists of extracellular glycoproteins and also contains digestive enzymes. The surface area of these cells is increased through the presence of microvilli to allow for the digestion and transport of molecules from the intestinal lumen. L-glutamate is a very important oxidative substrate for the intestinal mucosa and is needed in the biosynthesis of the two essential amino acids proline and arginine. L-glutamate is a key factor responsible for protection of the mucosa and dietary glutamate appears to be an essential factor for the maintenance of mucosal health and functions. Furthermore, glutamate is one of the most important neurotransmitters needed for normal brain function and nearly all excitatory neurons in the central nervous system are glutamatergic. In addition, it is estimated that over half of all brain synapses release this amino acid

    Glutamate plays an especially important role in clinical neurology because elevated concentrations of extracellular glutamate, released as a result of neural injury, are toxic to neurons. Free glutamic acid cannot cross the blood-brain barrier in the needed quantities but is instead converted into L-glutamine. The brain uses L-glutamine for fuel and protein synthesis. Glutamate is involved in cognitive functions like learning and memory in the brain. The neurotransmitter L-glutamate functions as an excitatory neurotransmitter and is a precursor for the synthesis of gamma-aminobutyric acid or γ-aminobutyric acid (GABA) in GABAergic neurons. GABAergic neurons transmit or secret γ-aminobutyric acid. L-Glutamate is also considered to be nature's "Brain food." L-glutamate helps to improve the mental capacities in humans and to speed up the healing of ulcers. In addition, L-glutamate has been reported to help control alcoholism, schizophrenia and the craving for sugar. However, excessive amounts of L-glutamate may cause neuronal damage and are associated with diseases such as amyotrophic lateral sclerosis, lathyrism, and Alzheimer's disease. Glutamate activates both ionotropic and metabotropic glutamate receptors. Ionotropic and metabotropic receptors are both ligand gated transmembrane proteins. Ionotropic receptors change shape when they are bound by a ligand. This change in shape creates a channel that allows ions to flow through. On the other hand, metabotropic receptors do not have channels and activate a G-protein that in turn activates a secondary messenger that may then activate a "secondary effector" whose effects depend on the particular secondary messenger system.

    Glutamate can cause neuronal damage and eventual cell death, particularly when N-Methyl-D-aspartic acid or N-Methyl-D-aspartate (NMDA) receptors are activated. High dosages of glutamic acid may induce symptoms such as headaches and neurological problems. The pathological process called excitotoxicity damages and kills nerve cells. This is caused via excessive stimulation by neurotransmitters such as glutamate and similar substances. Unfortunately, glutamate is of transient nature in biological systems and is therefore extremely difficult to study in action. However, quantitative amino acid analysis can be used to determine the presence of L-glutamate in different tissue types, plasma and other bodily fluids including dietary supplements and foods.

    References:

    1. Augustin H, Grosjean Y, Chen K, Sheng Q, Featherstone DE: Nonvesicular release of glutamate by glial xCT transporters suppresses glutamate receptor clustering in vivo. J Neurosci. 2007 Jan 3; 27(1):111-23.
    2. Burrin DG, Janeczko MJ, Stoll B.; Emerging aspects of dietary glutamate metabolism in the developing gut. Asia Pac J Clin Nutr. 2008; 17 Suppl 1:368-71.
    3. Corrie JE, DeSantis A, Katayama Y, Khodakhah K, Messenger JB, Ogden DC, Trentham DR: Postsynaptic activation at the squid giant synapse by photolytic release of L-glutamate from a ‘caged’ L-glutamate. J Physiol. 1993 Jun; 465:1-8.
    4. D’Mello, J.P.F.; Amino Acids in Human Nutrition and Health. Stylus Pub Llc. ISBN-13: 9781845937980, ISBN-10: 1845937988
    5. Herrling, Paul L.; Excitatory Amino Acids, 1st Edition. Clinical Results with Antagonists. 29 Jan 1997, Academic Press. ISBN : 9780125468206, eBook ISBN : 9780080531342. Mar 2012. ISBN-13: 9781845937980. ISBN-10: 1845937988
    6. Journal of amino acids: http://www.ncbi.nlm.nih.gov/pmc/journals/1711/ , and, http://www.hindawi.com/journals/jaa/
    7. Okumoto S, Looger LL, Micheva KD, Reimer RJ, Smith SJ, Frommer WB: Detection of glutamate release from neurons by genetically encoded surface-displayed FRET nanosensors. Proc Natl Acad Sci U S A. 2005 Jun 14;102(24):8740-5. Epub 2005 Jun 6.
    8. Reeds PJ, Burrin DG, Stoll B, Jahoor F: Intestinal glutamate metabolism. J Nutr. 2000 Apr;130(4S Suppl):978S-82S.
    9. Smith QR: Transport of glutamate and other amino acids at the blood-brain barrier. J Nutr. 2000 Apr;130(4S Suppl):1016S-22S.

     


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  • 11/20/13--00:00: Taurine
  • Taurine, also called 2-aminoethanesulfonic acid, is a sulfonic acid of the formula HOSO2-CH2-CH2-NH2 and has a molecular weight of 125.15 g mol−1 that is widely distributed in animal tissues. It is also characterized as an amino acid. The presence of a sulfonic group gives taurine a pKa value of 1.5 making it the most acidic amino acid. Taurine is not incorporated into proteins but is found in high concentrations in animals, including insects and arthropods, however, taurine is generally absent or only present in traces in bacteria and plants. It is one of the most abundant low-molecular-weight organic molecules present in animals. Huxtable in 1992, reported that a 70-kg human contains up to 70 g of taurine. Furthermore, taurine is found in bile and the large intestine. Taurine was first isolated from the bile of the ox, Bos Taurus, which gave it its name. Taurine is known to have many fundamental biological roles such as conjugation of bile acids, antioxidation, osmoregulation, membrane stabilization and modulation of calcium signaling, and it is essential for cardiovascular function, the development and function of skeletal muscle, the retina and the central nervous system. Taurine plays a protective role against free radicals and toxins in various cells and tissues. One of the reported functions of taurine is to protect cells against oxidation, by protecting mitochondrial integrity and respiration. However, many functions of taurine appear to be still unknown. It is now thought that taurine plays an important role in human nutrition. Furthermore, there is also indication that taurine may delay the aging process when taken as a supplement. Taurine appears to scavenge free radicals and its useful effects are attributed to its capability to stabilize biomembranes, to scavenge reactive oxygen species (ROS), and to decrease the peroxidation of unsaturated membrane lipids. Taurine appears to be part of the antioxidant defense systems found in mammalian cells. These systems consist of nonenzymatic antioxidants with low molecular weights including the vitamins A and E, betacarotene, uric acid, and enzymes such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), as well as glutathione reductase (GR). During the metabolism of sulphur-containing amino acids taurine is synthesized from its precursors methionine and cysteine therefore this amino acid is considered to be non-essential or conditionally essential. A clear toxicity has not been established for taurine yet but no adverse affects have been observed for any dose tested so far. Uozumi et al., in 2006, reported that the taurine transporter gene (TauT) is up-regulated during muscle differentiation. These results suggest that the taurine/TauT system provides resistance to muscle degeneration or wasting. Shao and Hathcock, in 2007, report that for doses of up to 3 g per day no side effects or toxic effects have been found. Therefore an upper dose of 3 g per day was selected for taurine supplementation as the “observed safe level” (OSL). The safety of taurine intake has been investigated since taurine is present in many energy drinks. The European Food Safety authority (EFSA) has concluded that taurine does not present any safety concerns for the levels currently used in energy drinks. In addition, a study performed in rats indicated that taurine may delay the aging progress. Diets high in meat and seafood will provide a higher intake than a vegetarian diet. Since plants contain very little taurine vegetarian may need to supplement it in addition to their vegetarian diet.

    Bio-Synthesis offer quantitative amino acid analysis for taurine supplment. This method can be used for the analysis of taurine in different tissue types, plasma and other bodily fluids including dietary supplements and foods. Contact us at 800.227.0672 or write to us at info@biosyn.com.

    Reference:

    1. Deng X, Liang J, Lin ZX, Wu FS, Zhang YP, Zhang ZW. Natural taurine promotes apoptosis of human hepatic stellate cells in proteomics analysis. World J Gastroenterol 2010;16(15): 1916-1923.
    2. Karl-Erik Eilertsen, Rune Larsen, Hanne K. Mæhre, Ida-Johanne Jensen and Edel O. Elvevoll; Anticholesterolemic and Antiatherogenic Effects of Taurine Supplementation is Model Dependent. In Biochemistry, Genetics and Molecular Biology » "Lipoproteins - Role in Health and Diseases", book edited by Sasa Frank and Gerhard Kostner, ISBN 978-953-51-0773-6, Chapter 11.
    3. Marit Espe, Elisabeth Holen; Taurine attenuates apoptosis in primary liver cells isolated from Atlantic salmon (Salmo salar). British Journal of Nutrition 2013-07-01. PMID 23182339.
    4. Huxtable RJ.; Physiological actions of taurine. Physiol Rev. 1992 Jan;72(1):101-63.
    5. Miyazaki T, Bouscarel B, Ikegami T, Honda A, Matsuzaki Y.; The protective effect of taurine against hepatic damage in a model of liver disease and hepatic stellate cells. Adv Exp Med Biol. 2009;643:293-303. doi: 10.1007/978-0-387-75681-3-30.
    6. Yoriko Uozumi, Takashi Ito, Yuki Hoshino, Tomomi Mohri, Makiko Maeda, Kyoko Takahashi, Yasushi Fujio, and Junichi Azuma; Myogenic differentiation induces taurine transporter in association with taurine-mediated cytoprotection in skeletal muscles. Biochem J. 2006 March 15; 394(Pt 3): 699–706.


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  • 11/20/13--00:00: Doxorubicin Conjugates
  • Doxorubicin is an anthracycline antibiotic used in cancer chemotherapy, which is also being evaluated as an antiviral agent. Two different mechanisms have been proposed to explain doxorubicin anticancer activity, in one it intercalates into the DNA and disrupts the topoisomerase-II DNA repair, leading to DNA-strand breaks at certain sites which are specific for doxorubicin; in the other mechanism, the antibiotic generates free radicals that damage cell membranes, DNA and proteins. Yet, the evidence supports the mechanism of poisoning the topoisomerase-II as the main anti-cancer mechanism for this drug. While therapeutically effective in a variety of tumor cells, doxorubicin is cardiotoxic and causes life-threatening hearth damage. Thus, to minimize this drug’s side-effects, it is usually administered as a conjugate, where the drug is covalently linked to another molecule that targets cancer cells. These various molecules include monoclonal antibodies, peptides, oligonucleotides and different natural and synthetic polymers.

    In the case of mAbs, the drug can be conjugated to either the whole antibody or its Fab fragment that is specific for some tumor associated antigen and that upon binding to that antigen it is internalized in the cell. Once inside the cell, the conjugate’s linker is cleaved, depending on the linker, either enzymatically or by a change in pH in certain cell compartments, usually the endosome/lysosome, releasing the drug inside the cell. The number of drug molecules attached per antibody may be increase by using polymeric linkers or carriers, this way increasing the cytotoxic effects of the conjugate. Doxorubicin can be also conjugated to certain cyclic or linear peptides in order to enhance cellular uptake and cellular retention, lessening the efficient drug efflux pump of certain cancer cells. In this kind of conjugates, the peptide moiety in addition to acting as a targeting agent would also be a factor in the intracellular drug retention, depending on the time to be degraded intracellularly.

    Doxorubicin can also be conjugated to another small molecule, like folic acid, to recognize and bind to folate receptor, which are over expressed in certain tumor cells. By using as linkers small polyethylene glycols, these conjugates can form micelles where the folic acid is exposed to the cell surface, whereas the drug is sequestered in the micelles’ interior. This way the delivery of drug per cell is significantly magnified. Doxorubicin can also be conjugated to small DNA or RNA fragments called aptamers; like antibodies, these oligonucleotides can be screen for their affinity with specific receptors and use them as the targeting moiety of the conjugate. Increased delivery of the drug to cells may be achieved by using a polysaccharide, such as dextran or pullulan, as a scaffold to which a ligand for a cell receptor and doxorubicin are linked covalently; a type of construct that increases the therapeutic benefits of the drug, while minimizing its side effects.

    Contact Bio-Synthesis for your doxorubicin drug bioconjugation.


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    DNA Methylation Analysis Techniques in Epigenetics

    By Klaus D. Linse

    After sequencing the human genome scientists began to realize that just knowing the genetic information of an individual is not sufficient to understand its phenotype. The way a DNA sequence is translated into function does not directly depend on the sequence itself. It has now become clear that the interaction of genes with environmental factors plays a role as well. The new field of epigenetics attempts to integrate the different chemical interactions or languages that genomes and environment use to communicate with each other. Chemically, epigenetics can be described as structural adaptations of chromosomal regions that reflect silenced or activated states of genes in response of the epigenetic signaling network of a genome to environmental factors. DNA methylation, indicating a chemical epigenetic change or imprinting, is the most widely studied modification. The modification is found within CpG dinucleotides. DNA methylation is a biochemical process where a DNA base, usually cytosine, is enzymatically methylated at the 5-carbon position. This epigenetic modification is associated with gene regulation, and is of paramount importance to biological health and disease. Over the years a growing number of chemical modification at different amino-acid residues mostly located at histone tails have been identified as well. Examples are acetylation, methylation, phosphorylation, ubiquitination and sumoylation, and new types of modifications are still waiting to be discovered. Other epigenetically important features are factors like nuclear positioning, noncoding RNAs, and microRNAs, among others, which are also associated with gene regulation and chromatin structure. In biochemical and biological science the speed of discovery of novel findings goes hand in hand with the development of new techniques and/or scientific instrumentation. The past few years have witnessed the introduction of a whole host of new techniques that allow for the characterization of the epigenome. Specifically, the number of studies that attempt to characterize the epigenome of normal or altered genetic states has grown exponentially. The following tables outline a list of techniques and methods as well as current microarray platforms that have been developed for the study of the epigenome and their applications in biology and medicine.

    Table 1: A List of techniques Used for Genome-Wide DNA Methylation Analyses
    Method Principle Reference
    RLGS
    (restriction landmark genomic scanning)
    Methylation-sensitive restriction digestion and 2D electrophoresis Kawai, J. et al.;Methylation profiles of genomic DNA of mouse developmental brain detected by restriction landmark genomic scanning (RLGS) method, Nucleic Acids Res, 21, 5604-8, 1993.
    MCA or MCAM
    (restriction landmark genomic scanning)
    Methylation-sensitive restriction digestion, printed membranes and dot-blot analysis or microarray hybridization Estecio, M.R. et al.; High-throughput methylation profiling by MCA coupled to CpG island microarray, Genome Res, 17, 1529-36, 2007.Ibrahim, AE. et al.; MMASS: an optimized array based method for assessing CpG island methylation, Nucleic Acids Res, 34, 36, 2006.
    DMH
    (differential methylation hybridization)
    Methylation-sensitive restriction digestion and microarray hybridization Huang, T.H. et al.; Methylation profiling of CpG islands in human breast cancer cells, Hum Mol Genet,8,459-70, 1999.
    CpG Island Array Amplicons, representing a pool of methylated CpG DNA derived from these samples, were used as hybridization probes in an array panel containing 1104 CpG island tags. Pearlly s. Yan, Martin R. Perry, Douglas E. Laux, Adam L. Asare, Charles W. Caldwell, and Tim Hui-Ming Huang; CpG Island Arrays: An Application toward Deciphering Epigenetic Signatures of Breast. Cancer Clin Cancer Res April 2000 6; 143.
    AIMS
    (amplification of inter-methylated sites)
    Methylation-sensitive restriction digestion, ID electrophoresis Frigola, J. et al.; Methylome profiling of cancer cells by amplification of inter-methylated sites (AIMS), Nucleic Acids Res, 30, e28, 2002.
    MSO microarray
    (methylation-specific oligonucleotide)
    Bisulfite conversion, PCR, bead array hybridization Gitan, R.S. et al.; Methylation-specific oligonucleotide microarray: a new potential for high-throughput methylation analysis, Genome Res, 12, 158- 64, 2002.
    ChIP-on-chip Chromatin immunoprecipitation with antibodies against MBDs and microarray hybridization Ballestar, E. et al.; Methyl-CpG binding proteins identify novel sites of epigenetic inactivation in human cancer, Embo J, 22, 6335-45, 2003.
    NotI digestion coupled to BAC array Methylation-sensitive restriction digestion and microarray hybridization Ching, T.T. et al.; Epigenome analyses using BAC microarrays identify evolutionary conservation of tissue-specific methylation of SHANK3, Nat Genet, 37, 645-51, 2005.
    MeDIP-on-chip Isolation by 5-methylcytosine antibody and microarray hybridization.Immunocapture followed by DNA microarray analysis to generate methylation profiles of all human chromosomes at 80-kb resolution. Weber, M. et al.; Chromosome-wide and promoter specific analyses identify sites of differential DNA methylation in normal and transformed human cells, Nat Genet, 37, 853-62, 2005.
    MClp-on-chip
    (methyl- CpG immunoprecipitation)
    Isolation by MBD-Fc beads and microarray hybridization. Gebhard, C. et al.; Genome-wide profiling of CpG methylation identifies novel targets of aberrant hypermethylation in myeloid leukemia, Cancer Res, 66, 6118-28, 2006.
    HELP assay
    (Hpall tiny fragment Erichment by Ligation- mediated PCR)
    Methylation-sensitive restriction digestion plus microarray hybridization.Intragenomic profiling and intergenomic comparisons of cytosine methylation. Khulan, B. et al., Comparative isoschizomer profiling of cytosine methylation: the HELP assay, Genome Res, 16, 1046-55, 2006.
    Methylation-specific bead arrays Bisulfite conversion, allele-specific primer extension, and bead array hybridization Bibikova, M. et al.; High-throughput DNA methylation profiling using universal bead arrays. Genome Res, 16, 383-93, 2006.
    MSNP
    (single nucleotide polymorphism chip-based method for profiling DNA methylation)
    Methylation-sensitive restriction digestion and SNP-chip hybridization Yuan, E. et al.;, A single nucleotide polymorphism chip-based method for combined genetic and epigenetic profiling: validation in decitabine therapy and tumor/normal comparisons, Cancer Res, 66, 3443-51 , 2006.
    MMASS
    (Combining array-based assays)
    Combinations of methylation-sensitive restriction digestions plus microarray hybridization Nielander, I. et al.; Combining array-based approaches for the identification of candidate tumor suppressor loci in mature lymphoid neoplasms, APMIS 115, 1107-34, 2007.
    MIRA-Assisted Microarray Analysis (methylated-CpG island recovery assay) Isolation of methylated DNA by affinity to the MBD2/MBD3Ll complex plus microarray hybridization Rauch, T. et al.; MIRA-assisted microarray analysis, a new technology for the determination of DNA methylation patterns, identifies frequent methylation of homeo domain containing genes in lung cancer cells, Cancer Res, 66, 7939-47, 2006.
    MSDK (Methylation- specific digital karyotyping) Methylation-sensitive restriction digestion plus SAGE Hu, M. et al.; Methylation-specific digital karyotyping, Nat Protoc, 1, 1621-36, 2006.
    aPRIMES
    (Array-based profiling of reference- independent methylation status)
    Differential restriction and competitive hybridization of methylated and unmethylated DNA Pfister, S. et al.; Array-based profiling of reference independent methylation status (aPRIMES) identifies frequent promoter methylation and consecutive downregulation of ZIC2 in pediatric medulloblastoma, Nucleic Acids Res, 35, e51, 2007.
    Expression profiling after demethylation Treatment with demethylating agents and expression Microarray in cells with and without treatment Suzuki, H. et al.; A genomic screen for genes upregulated by demethylation and histone deacetylase inhibition in human colorectal cancer, Nat Genet, 31,141-9, 2002.
    MTA
    (methylation target array)
    In MTA, linker-ligated CpG island fragments were digested with methylation-sensitive endonucleases and amplified with flanking primers Chuan-Mu Chen, Hsiao-Ling Chen, Timothy H.-C. Hsiau, Andrew H.-A. Hsiau, Huidong Shi, Graham 1. R. Brock, Susan H. Wei, Charles W. Caldwell, Pearlly S. Yan, and Tim Hui-Ming Huang; Methylation Target Array for Rapid Analysis ofCpG Island Hypermethylation in Multiple Tissue Genomes. Am J Pathol. 2003 July; 163(1): 37-45.
    Table 2: Current Microarray Platforms Used for Epigenomic Studies
    Microarray Platform for Epigenomics Resolution Number of C Iones/Oligos* Coverage
    BAC/PAC clones 100-200 kb Up to ~33,000 ~ Complete genome
    CpG islands 100-1000 bp Up to ~ 12,000 CpG islands
    Oligonucleotides      
    Promoter 25-60 bp** 244,000;385,000; 4.6 million Promoter regions
    CpG island 25-60 bp** 244,000; 385,000 CpG islands
    Tiling 25-60 bp** Up to 45 million (set of 7 arrays) ~ Complete genome
    CpG-dinucleotide specific 1 bp Up to 1536 Selected promoters
    * Improved microarrays with higher resolution are constantly being developed, so the number of oligos on a single array increases as new platforms become available.
    ** This is the size of the oligonucleotide; the final resolution depends on the method used to enrich the DNA for methylated sequences or histone modifications.

    CHROMATIN IMMUNOPRECIPITAION

    CHROMATIN IMMUNOPRECIPITAION is a technique were intact nuclei are gently fixed to maintain the physical relationship of DNA-binding molecules to genomic DNA. The chromatin (DNA plus bound molecules) is sheared to small fragments and incubated with an antibody that selectively immuno-precipitates one of the bound molecules. The binding sites of the molecule (usually a protein) of interest become apparent from their enrichment in the immune-precipitated fraction of the genome.

    Table 3: Early non-specific and differential gene methylation analysis methods
    Early non-specific method Early differential methods
    Restriction endonuclease digestion, isotope incorporation, and TLC Isoschizomer digestion and isotope incorporation
    Polyclonal leporine antibody, radiolabeled DNA HpaII PCR
    RP-HPLC Methylation-specificRLGS
    HPLC, mass spectrometry AP-PCR
    SssI methyltransferase tritium labeling AIMS
    Monoclonal, isothiocyanate labeled fluorescent anti-5mC  

    Legend:TLC, thin-layer chromatography; RP-HPLC, reverse phase high performance liquid chromatography; Anti-5mC, anti-5-methylcytosine; RLGS, restriction landmark genome scanning; AP-PCR, arbitrarily primed polymerase chain reaction; AIMS, amplification of intermethylated sites.

    Table 4: Other Methods
    Sodium Bisulfate Treatment Microarray Technologies
    Ligation-mediated PCR Methyl-sensitive restriction enzymes; CGI library
    Bisulfite sequencing Anti-methylcytosine immune precipitation; SMRT
    MS-PCR Anti-methylcytosine immunoprecipitation; Promoter array
    MS-SNuPE Methyl-binding protein precipitation; CGI library
    MS-SSCA Methyl-binding protein precipitation; CGI library
    MS-HRM Sodium bisulfate treatment; Oligonucleotides
    Bisulfite treatment to create new restriction sites Sodium bisulfate treatment Illumina; beadchip

    Legend:MS-PCR, methylation-specific polymerase chain reaction; MS-SNuPE, methylation-specific single nucleotide primer extension; MS-SSCA, methylation- specific single-strand conformation analysis; MS-HRM, methylation-specific high resolution melting; CGI, CpGislandmicroarray; SMRT,submegabase resolution tiling array; MeDIP,methylated DNA immunoprecipitation; MeCIP,methyl-CpG immunoprecipitation; Oligonucleotides, Whole genome oligonucleotide array.

    Table 5: DNA methylation analyses by next-generation sequencing.
    Method Genome coverage
    Bisulfite sequencing Whole genome
    MeDIP-seq1 anti-5mC Enriched Methylated DNA
    MBDiGS2 Enriched Methylated DNA
    MRE-seq3 Size selected fraction
    MMSDK4 Representative genome tags

    1 Sequencing of immunoprecipitated anti-5mC DNA

    2 Methyl-binding protein precipitated sequencing.

    3 Methyl-sensitive restriction enzyme sequencing.

    4 Modified methylation-specific digital karyotyping.

    Time line of DNA methylation analysis

    Harrison & Parle-McDermott published a paper in 2011 describing the speed in the development of new techniques to study DNA methylation. The following figure illustrates this quite nicely.

    Time line of DNA methylation analysis

    Time line of DNA methylation analysis. Early techniques used in the 1980s allowed to measure the amount of 5-methylcytosine within a particular genome. Since then a variety of methods have been developed that allow for a more detailed study of the epigenome. These new type of methods or assays include the use of methylation-sensitive restriction enzymes, immunoprecipitation, bisulfite sequencing, usually in combination with the polymer chain reaction (PCR), the use of microarrays, reversed-phase high-performance liquid-chromatography (RP-HPLC), methylation-sensitive single nucleotide primer extension (MS-SnuPe), combined bisulfate restriction analysis (COBRA), arbitrarily primed PCR (APPCR), amplification of inter-methylated sites (AIMS), reduced representation bisulfite sequencing (RRBS), and finally next-generation sequencing, to name a few.

    Future of Methylome Analysis

    It is expected that next-generation sequencing approaches for DNA methylation analysis will dominate for a while. Newer sequencing technologies, such as single-molecule real-time sequencing (SMRT) are needed to directly detect all known DNA methylation reactions without the need for bisulfate treatment. As pointed out by Harrison and Parle-McDermott the major developments in the methodologies for profiling and fingerprinting the human methylome have followed a clear progression toward innovative sequencing techniques that allow for single-pair resolution. It is expected that as the technologies improve the cost of genome-wide sequencing will decrease. This will result in new waves of data and the need for better bioinformatics tools to allow for the accurate analysis of vast datasets in the coming years.

    Reference

    Weixing Feng, Zengchao Dong, Bo He and Kejun Wang; Analysis method of epigenetic DNA methylation to dynamically investigate the functional activity of transcription factors in gene expression. BMC Genomics 2012, 13:532 doi:10.1186/1471-2164-13-532.

    Alan Harrison and Anne Parle-McDermott; DNA Methylation: A Timeline of Methods and Applications Front Genet. 2011; 2: 74.)

    Lanlan Shena and Robert A. Waterland; Methods of DNA methylation analysis. Curr Opin Clin Nutr Metab Care 10:576–581. 2007 Lippincott Williams & Wilkins.


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  • 11/22/13--00:00: ABT-510 Peptide
  • Bio-Synthesis provides ABT-510 peptide syntehsis, this synthetic peptide that mimics the anti-angiogenic activity of the endogenous protein thrombospondin-1 (TSP-1). The systematic name for the ABT-510 peptide is N-Acetyl-N-methylglycylglycyl-L-valyl-D-isoleucyl-L-threonyl-L-norvalyl-L-isoleucyl-N5-(diaminomethylene)-L-ornithyl-N-ethyl-L-prolinamide with the molecular formula C46H83N13O11, an average mass of 994.231689 Da, a monoisotopic mass of 993.633484 Da and the ChemSpider ID 5293759. ABT-510 is an antiangionic TSP-1 modified nonapeptide that was designed using the 7-mer active sequence GVITRIR of the second type I repeat as the target sequence. ABT-510 inhibits the formation of new blood vessels. Furthermore, ABT-510 inhibits the actions of several pro-angiogenic growth factors important to tumor neovascularization. In addition, the modified TSR peptide ABT-510 inhibits malignant glioma growth in vivo and induces apoptosis of brain microvessel endothelial cells (MvEC) propagated in vitro. ABT-510 also increases active TGF-1 levels in tumors. The term “neovascularization” describes the proliferation of blood vessels in tissue not normally containing them or the proliferation of blood vessels of a different kind than usual present in tissue. These pro-angiogenic growth factors include vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), and interleukin 8 (IL-8). Thrombospondin 1 (TSP-1) is a large multifunctional glycoprotein involved in multiple biological processes including angiogenesis, apoptosis, and activation of TGF-1. ABT-510 is a TSP-1 synthetic analog that mimic its antiangiogenic action. Tumors that over express TSP-1 grow more slowly, have fewer metastases, and decreased angiogenesis. Therefore, TSP-1 provides a novel target for the treatment of cancer.

    ABT 510 Peptide Synthesis

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  • 11/25/13--00:00: Nanoparticle Applications
  • Nanoparticles in Biology and Medicine

    A brief history of firsts in nanotechnology: Nanotechnology started in 1959 when Feynman gave an after-dinner talk describing molecular machines built with atomic precision. In 1974, Taniguchi used the term "nano-technology" in a paper on ion-sputter machining. The molecular nanotechnology concept was coined by Drexler at MIT in 1977. The first technical paper on molecular engineering to manufacture with atomic precision was published in 1981, when the scanning tunneling microscope (STM) was invented. The Buckyball was discovered in 1985, and the first book on nanotechnology was published in 1986. During the same year, atomic force microscopy (AFM) was invented, and the first nanotechnology organization was formed. The first protein was engineered in 1987, and many firsts followed in nanotechnology, including the publication of the first textbook about the field in 1992, as well as the first nanomedicine book published in 1999. And it all starts with particles.

    Let us find out what a particle is. A particle is a small, localized object that behaves as a whole unit and can be described using physical properties such as volume, size and mass. A nano-particle is "a particle having one or more dimensions of the order of 100 nm or less." Usually, fine particles range in their size between 2,500 and 100 nanometers. However, ultrafine particles, or nanoparticles, range in sizes between 100 and 1 nanometers.

    During the 1970-80’s, when the first thorough fundamental studies were running with "nanoparticles" in the United States (by Granqvist and Buhrman) and Japan, (within an ERATO Project), they were called "ultrafine particles"(UFP). During the 1990s, before the National Nanotechnology Initiative was launched in the United States, the new name, "nanoparticle" had become fashionable. Nanoclusters have at least one dimension between 1 and 10 nanometers and a narrow size distribution. Nanopowders are agglomerates of ultrafine particles, nanoparticles, or nanoclusters. Nanometer-sized single crystals or single-domain ultrafine particles, are often referred to as nanocrystals. The field of nanotechnology is rapidly developing, and new types of nanomaterials are being developed constantly. It is expected that nanomaterials will be developed at several levels, as part of material devices and systems. Many nanomaterials are now commercially available. The cells of living organisms are typically 10 μm in size. Parts of the cells are even much smaller, in the sub-micron size range.

    Figure 1: This figure illustrates the size of a nanoparticle. If a nanoparticle was the size of a football - this image shows how atoms, cells and organisms would compare at a more familiar scale to humans.

    Proteins that make up the cells nanomachinery are just around 5 nanometers (nm) in size. These are the sizes of the smallest man-made nanoparticles. Their size allows them to be used as probes to study the cells’ biological processes. Typically, for biological applications, nanomaterials are selected for their optical and magnetic properties. However, nanomaterials are also applied for novel electronic, optoelectronis, and memory devices. Figure 1 illustrates the size of nanoparticles.

    The fact that nanoparticles exist in the same size domain as proteins allows them to be used to label or tag proteins, either in vivo or in vitro. To allow the interaction of nanoparticles with the biological target, a molecular coating or layer acting as an interface will need to be attached to the particle. Examples of biological coatings include antibodies, biopolymers such as collagen, or monolayers of small biocompatible molecules. Since the use of optical detection techniques are widely used in biological research, nanoparticles should either fluoresce or change their optical properties in response to a biological function. Figure 2 illustrates the sizes and shapes of a few nanoparticles. A list of applications involving nanoparticles follows below.

    Sizes and shape of different nanoparticles

    Figure 2: Sizes and shape of different nanoparticles. This cartoon shows the morphology of nanoparticles that can be used for neuroimaging. Source: Moresco & Masserine 2012.

    Some applications of nano-materials in biology and medicine are:

    Fluorescent biological labels for fluorescent signaling
    Drug and gene delivery
    Bio detection of pathogens
    Detection of proteins – Antigen detection
    Probing of DNA structure
    Tissue engineering
    Tumor destruction via heating (hyperthermia)
    Separation and purification of biological molecules and cells
    Linker activated nanoparticles
    Biocompatible nanoparticles
    MRI contrast enhancement
    Molecular imaging such as computed tomography (CT), positron emission computed tomography (PET), single photon emission computed tomography (SPECT), and magnetic resonance (MRI),
    Phagokinetic studies

    Representation of multifunctional iron Figure 3: Representation of multifunctional iron oxide nanoparticles showing multiple modes of functionalization. Representation of multifunctional nanoparticlesFigure 4: Representation of multifunctional nanoparticles.

    Nanoparticles that usually form the core of nano-biomaterials can be composed of inorganic, polymeric materials or can be in the form of nano-vesicles surrounded by a membrane or a layer. The shapes of these particles can come in different morphologies. Furthermore, nanoparticles can be functionalized in multiple ways. Figures 3 and 4 show the graphical representation of different ways to functionalize nanoparticles. For example, as depicted in figure 3, the particles can be conjugated to different types of molecules, such as antibodies, green fluorescent protein (GFP), avidin, or streptavidin, as well as to DNA/RNA oligomers and gold particles or, as shown in Figure 4, contrast agents useful for CT/MRI imaging, radiotracers useful for PET/SPECT imaging, functionalized with drugs for targeted drug delivery or specific ligands, as well as special surfaces such as hydrophilic surfactants to enhance biocompatibility.

    It is thought that nanoparticles will play an increasing role in nanomedicine in the future. Nanomedicine applies nanotechnology with the goal to improve the quality of human lives. Useful medical applications of nanoparticles include improved drug delivery, such as protein, peptide, and oligonucleotide delivery in biological systems, nanoparticles to specific targets in tumors and cancers, and nanoparticles for tissue visualization to enhance surgical techniques or to visualize tumors. It is hoped that it will become possible in the near future to design nanorobots or nanomachines that allow for the repair of damage parts of the cell.

    More recently, many companies have begun to use nanotechnologies. The majority of the companies are small recent spinouts of various research institutions. Most of the companies are developing applications for the pharmaceutical industry, mainly to enhance or enable drug delivery. Other companies exploit quantum size effects in semiconductor nanocrystals to tag biomolecules or use gold nanoparticles for the labeling of various cellular parts. Cytodiagnostics, one such company, provides gold, silver, and magnetic nanoparticles with sizes ranging from 5 nm to 400 nm. These particles can be conjugated to biomolecules such as antibodies, BSA, KLH, and many others.

    Biosynthesis Inc. in Lewisville, Texas offers custom conjugation of antibodies or other proteins to this type of nanoparticles. To be able to perform custom conjugations, approximately 3 mg of purified and lyophilized antibody (or any other protein) is required. A successful conjugation results in a typical yield of 10 ml of protein-gold nanoparticles conjugated at an optical density of 3 (OD=3). This amount is enough to probe approximately 100 dot blot strips using 15 ml of a 1:100 diluted conjugate per strip.

    Services include: 1. antibody or protein sourcing, if needed; 2. conjugation of a customer protein to selected gold nanoparticles; and 3. Purification of the conjugate.

    Furthermore, oligonucleotides and other molecules can be conjugated to gold nanoparticles as well. Biosynthesis provides custom conjugation of single-stranded or double-stranded oligonucleotides to gold nanoparticles with sizes ranging from 5 nm to 400 nm. A successful conjugation typically results in a yield of 30 ml of oligonucleotide-gold nanoparticle conjugate at an optical density of 1 (OD=1).

    Services include: 1. synthesis of oligonucleotides for conjugation. The customers usually supplies the nucleotide sequence, and decides which terminal (5' or 3') will be attached to the gold surface, and Biosynthesis Inc. does the rest; 2. Conjugation of the oligonucleotide to a gold nanoparticle size of customer’s choice; 3. Purification of the conjugate.

    The following table shows a list of nanomedical technologies.

    Table-1: Nanomedicine Technologies (Source: Freitas 2005)
    Raw nanomaterials Cell simulations and cell diagnostics Biological research

    Nanoparticle coatings
    Nanocrystalline materials

    Cell chips
    Cell simulators

    Nanobiology
    Nanoscience in life sciences

    Artificial binding sites DNA manipulation, sequencing, diagnostics Drug delivery

    Artificial antibodies
    Artificial enzymes
    Artificial receptors
    Molecular imprinted polymers

    Genetic testing
    DNA microarrays
    Ultrafast DNA sequencing
    DNA manipulation and control

    Drug discovery Biopharmaceuticals
    Drug encapsulation
    Drug delivery
    Smart drugs

    Nanostructured materials Tools and diagnostics Biotechnology, biorobotics, and nanorobots

    Cyclic peptides
    Dendrimers
    Detoxification agents
    Fullerenes
    Functional drug carriers
    MRI scanning
    Nanobarcodes
    Nanoemulsions
    Nanofibers
    Nanoparticles
    Nanoshells
    Carbon nanotubes
    Noncarbon nanotubes
    Quantum dots

    Bacterial detection systems
    Biochips
    Biomolecular imaging
    Biosensors and biodetection
    Diagnostic and defense applications
    Endoscopic robots and microscopes
    Fullerene-based sensors
    Cellular imaging
    Lab on a chip
    Monitoring
    Nanosensors
    Point of care diagnostics
    Protein microarrays
    Scanning probe microscopy

    Biological viral therapy
    Virus-based hybrids
    Stem cells and cloning
    Tissue engineering
    Artificial organs
    Nanobiotechnology
    Biorobotics and biobots

    DNA-based devices and nanorobots
    Diamond-based nanorobots
    Cell repair devices

    Cytodiagnostics, another company, offers a unique proprietary protocol that produces particles with uniform shapes and a narrow size distribution. The gold nanoparticle surface can be modified to allow for the conjugation to molecules such as biotin and other molecules of choice. Furthermore, particles can be functionalized with carboxyl, amine, and methyl groups, among others.

    nanoparticles
    Figure 5: Sizes of gold nanoparticles are illustrated.
    Source: http://www.cytodiagnostics.com/gold_nanoparticle_products.php
    nanoparticles

    Silver Nanoparticles are also available with core sizes of 40 nm - 100nm

    Figure 6: Sizes of silver nanoparticles are illustrated.
    Source: http://www.cytodiagnostics.com/store/pc/Silver-Nanoparticles-c150.htm

    High-quality monodisperse silver nanoparticles with a narrow size distribution (CV <15%) are available as well. Nanosilver products are ideal for a wide array of applications such as “Conjugate Development,”, “Sensor Development,”, “Molecular Imaging’,’Surface Enhance Raman Spectroscopy (SERS),”, and“Bactericidal applications.”

    Adsorption spectrumFigure 6: Adsorption spectrum of silver nanoparticles in different wavelengths.
    Source: http://www.cytodiagnostics.com/store/pc/Silver-Nanoparticles-c150.htm

    Superparamagnetic iron nanoparticles, both in the metallic, and oxide forms, can be used for bioconjugation as well and are widely used in the life sciences. Typical applications include but are not limited to the separation and purification of biomolecules, such as proteins and DNA, from complex mixtures, as well as in immunoprecipitation protocols. By using only a magnet for fast pull-down and isolation of magnetic particles, cumbersome and long purification protocols using columns or centrifugation are not needed. Other applications may include medical diagnostics, catalysis, probing agents to indirectly study the structure of mixed self-assembled monolayers (SAMs), solid phase extraction, anode materials for Li-ion batteries, and electromagnetic interference (EMI) shielding materials.

    Source: http://www.cytodiagnostics.com/magnetic_nanoparticles.php
    Superparamagnetic iron nanoparticles

    The company Nanoprobes offers 1.4 nm Nanogold® particles. These are gold compounds that are not just adsorbed to proteins, like colloidal gold, but covalently reacts at specific sites under mild buffer conditions with molecules that are selected for conjugation. A well-defined product can be synthesized that can be purified chromatographically.

     Source: http://www.nanoprobes.com/products/LabRgts.html#feat

    Features of Nanogold®
    • Unparalleled penetration of conjugates up to 40 µm.
    • Higher density of immunolabeling than with larger gold probes.
    • Can be conjugated to any molecule with a suitable reactive group. Available with different reactivities.
    • Extremely uniform 1.4 nm gold particle
    • Label at specific sites, which do not obstruct native reactivity.
    • Close to stoichiometric labeling.
    • Reacts under mild, neutral conditions
    • Conjugates are easily isolated by gel filtration.
    • Conjugates are stable to a wide range of pH and ionic strengths.
    • High Representation of multifunctional ironstability: conjugates show unchanged reactivity after storage for a year.
    Nanogold
    ReferencesF Re, R Moresco and M Masserini; Nanoparticles for neuroimaging. Journal of Physics D: Applied Physics Volume 45 Number J. Phys. D: Appl. Phys. 45
    Robert A. Freitas Jr., What is nanomedicine? Nanomedicine: Nanotechnology, Biology, and Medicine 1 (2005) 2– 9 http://www.foresight.org/Nanomedicine/

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