Publication Date:
2012-08-23
Description:
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-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 structure–function relationships of modified nucleotides in dictating polymerase performance.
Print ISSN:
0305-1048
Electronic ISSN:
1362-4962
Topics:
Biology
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