Novel Biopolymers Based on an Expanded Genetic Alphabet
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چکیده
Structurally and functionally modified nucleotides expand the scope of therapeutic and diagnosticapplications of oligonucleotides. Incorporation of these into DNA and replication of oligonucleotidescontaining them by polymerases may cause pausing, early termination, and misincorporation,however. Reported here is a simple in vitro assay to screen for DNA polymerases that accept modified nucleotides, based on a set of primer extension reactions which, in combination with thescintillation proximity assay (SPATM), allows rapid and simple screening of multiple enzymes for theiraccuracy of replicating oligonucleotides in the presence of unnatural nucleotides. A proof of concept isobtained using pseudo-thymidine (\|/T; 1), the C-nucleoside analog of thymidine, as an unnatural substrate. The conformational properties of 1 arising from the carbon-carbon bond between the sugarand the base make it an interesting probe for the importance of conformational restraints in the activesite of polymerases during primer elongation. From a pool of commercially available thermostablepolymerases, the assay identified Taq DNA polymerase as the most suitable enzyme for the PCRamplification of oligonucleotides containing 1. Subsequent experiments analyzing processivity andfidelity of Taq DNA polymerase acting on 1 are presented.
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تاریخ انتشار 1999