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

ISSN： 1362-4962

Source： Nucleic Acids Research, Vol.40, Iss.15, 2012-08, pp. : 7404-7415

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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 45nt/s, comparable to the fastest NGS chemistries, yet with catalytic efficiencies (k _pol/k _D) comparable to natural nucleotides. Pre-steady-state kinetic studies of thymidine analogs revealed that the major determinant for improved nucleotide selectivity is a significant reduction in k _pol by >1000-fold over TTP misincorporation. These studies highlight the importance of structurefunction relationships of modified nucleotides in dictating polymerase performance.