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

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) methoddependsonefficientbase-selectiveincorporation of reversible terminators by DNA polymerases. Most terminators are designed with 3 0 -O-blocking groups but are incorporated with low efficiency and fidelity. We have developed a novel class of 3 0 -OH unblocked nucleotides, called Lightning Terminators TM , 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 TM DNA polymerase, we demonstrate that these 3 0 -OH unblocked terminators exhibit superior enzymatic performance compared to two other reversible terminators, 3 0 -O-amino-TTP and 3 0 -O-azidomethyl-TTP. Lightning Terminators TM show maximum incorporation rates (kpol )t hat range from 35 to 45nt/s, comparable to the fastest NGS chemistries, yet with catalytic efficiencies (kpol/KD) comparable to natural nucleotides. Pre-steady-state kinetic studies of thymidine analogs revealed that the major determinant for improvednucleotide selectivity is a significant reduction in kpol by >1000-fold over TTP misincorporation. These studies highlight the importance of structure–function relationships of modified nucleotides in dictating polymerase performance.