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Meet the Avecia-TriLink Alliance Team at EuroTIDES, Nov 17-20 in Barcelona.

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Research Update

Discovering Secondary & Tertiary RNA Structure

To fully understand RNA function, it is important to elucidate not only the nucleotide sequence, but the folding dynamics over time. The study of RNA folding kinetics provides insight into biological functions such as viral RNA replication, activation of open-reading frames and translation. A powerful method for probing RNA kinetics was described in a detailed protocol published by S. Mortimer and K. Weeks of UNC, Chapel Hill. SHAPE, or selective 2'-hydroxyl acylation analyzed by primer extension, makes possible quantitative, single-nucleotide resolution of RNA folding dynamics at a one second time resolution. During the SHAPE reaction, 2'-hydroxyl acylation with benzoyl cyanide modifies the flexible nucleotides. A cDNA synthesis of the resultant RNA allows for quantitative determination of the degree of modification at a given position along the RNA, where the most flexible nucleotides are the most highly modified. cDNA synthesis was perfomed using TriLink’s 2',3'-dideoxy-ATP, -CTP, -GTP and -TTP. This method shows strong potential to be a straight-forward solution for benchtop RNA folding kinetics investigations.

With advances in our synthesis protocols, our high quality ddNTPs are now available at a new lower price.

2',3'-Dideoxynucleotide Set

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Technology Spotlight

Understanding Drug Resistance Through Kinetics and Molecular Modeling

Several nucleotide analogue reverse transcriptase inhibitors are approved for treatment of chronic Hepatitis B Virus (HBV) infection. Entecavir (ETV) is a deoxyguanosine analog used in the treatment of HBV, which acts as a competitive inhibitor of HBV polymerase. A recent study by A. Walsh et. al. sought to determine the mechanism of ETV-resistance observed in Phase II trials. The goal of this work was to study the kinetics of nucleotide incorporation by wild-type HBV and ETV resistant mutants of HBV. For the in vitro experiments, ddGTP (Cat# N-4002) and the triphosphate version of ETV (ETV-TP, prepared by TriLink) were evaluated as HBV polymerase substrates. In this work, the kinetics of ETV-TP were studied as the intracellular phosphorylated form of the molecule produced in vivo, with ddGTP used as a control for competitive inhibition of HBV polymerase. Kinetic characterization in combination with molecular modeling suggest that the mechanism for ETV resistance is exclusion of ETV-TP from the dNTP binding site.

Specialized nucleic acid products are key tools in characterizing and modeling drug function. TriLink offers custom polyphosphate synthesis, including mono-, di- and triphosphates. We can phosphorylate your existing nucleoside precursor or prepare the starting nucleoside, even if there is currently no known synthesis scheme. Contact us to discuss your project today!

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Question of the Month

Q - Which modifications impart nuclease resistance?

A - Unmodified phosphodiester oligonucleotides are highly susceptible to degradation by intracellular and extracellular nucleases. The most common form of degradation comes from 3'-exonuclease activity. Fortunately, there are many different modifications that can increase resistance to nuclease degradation and therefore opportunity for therapeutic application. Phosphorothioate
/methylphosphonate backbone modifications and 2'-O-Methyl/2'-Fluoro sugar modifications can prevent both endo- and exonuclease degradation. TriLink offers the largest selection of alpha thio and 2'-O-Methyl/2'-Fluoro modified NTP’s available to allow preparation of nuclease resistant sequences.

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