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

• Published in: Nucleic acids research Vol. 40; no. 15; pp. 7404 - 7415
• Main Authors: Gardner, Andrew F, Wang, Jinchun, Wu, Weidong, Karouby, Jennifer, Li, Hong, Stupi, Brian P, Jack, William E, Hersh, Megan N, Metzker, Michael L
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 01.08.2012
• Subjects: Deoxyuracil Nucleotides - chemistry; Deoxyuracil Nucleotides - metabolism; DNA - biosynthesis; DNA - chemistry; DNA sequencing; DNA-directed DNA polymerase; DNA-Directed DNA Polymerase - chemistry; DNA-Directed DNA Polymerase - metabolism; Electricity; Fidelity; Genomics; High-Throughput Nucleotide Sequencing; Kinetics; Nitrobenzenes - chemistry; Nucleotide sequence; Nucleotides; Nucleotides - chemistry; Nucleotides - metabolism; Probes; Sequence Analysis, DNA; Structure-function relationships; Thymidine
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/gks330

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.