Rapid incorporation kinetics and improved fidelity of a novel class of 3'-OH unblocked reversible terminators.

Nucleic Acids Research
Andrew F GardnerMichael L Metzker

Abstract

Recent developments of unique nucleotide probes have expanded our understanding of DNA polymerase function, providing many benefits to techniques involving next-generation sequencing (NGS) technologies. The cyclic reversible termination (CRT) method depends on efficient base-selective incorporation of reversible terminators by DNA polymerases. Most terminators are designed with 3'-O-blocking groups but are incorporated with low efficiency and fidelity. We have developed a novel class of 3'-OH unblocked nucleotides, called Lightning Terminators™, which have a terminating 2-nitrobenzyl moiety attached to hydroxymethylated nucleobases. A key structural feature of this photocleavable group displays a 'molecular tuning' effect with respect to single-base termination and improved nucleotide fidelity. Using Therminator DNA polymerase, we demonstrate that these 3'-OH unblocked terminators exhibit superior enzymatic performance compared to two other reversible terminators, 3'-O-amino-TTP and 3'-O-azidomethyl-TTP. Lightning Terminators show maximum incorporation rates (k(pol)) that range from 35 to 45 nt/s, comparable to the fastest NGS chemistries, yet with catalytic efficiencies (k(pol)/K(D)) comparable to natural nucleotides. Pre-stea...Continue Reading

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Citations

Apr 8, 2015·Chembiochem : a European Journal of Chemical Biology·Zitian ChenYanyi Huang
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May 25, 2021·Computational and Structural Biotechnology Journal·Eojin YooByung-Kwan Cho

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Software Mentioned

PyMOL Molecular Graphics System
Peak Scanner
Vent
Therminator
MacPyMol

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