title: Rapid incorporation kinetics and improved fidelity of a novel class of 3'-OH unblocked reversible terminators.
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-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.
authors.affiliation: New England Biolabs, Ipswich, MA 01938, USA.
authors.forename: Andrew F
authors.initials: AF
authors.lastname: Gardner
authors.forename: Jinchun
authors.initials: J
authors.lastname: Wang
authors.forename: Weidong
authors.initials: W
authors.lastname: Wu
authors.forename: Jennifer
authors.initials: J
authors.lastname: Karouby
authors.forename: Hong
authors.initials: H
authors.lastname: Li
authors.forename: Brian P
authors.initials: BP
authors.lastname: Stupi
authors.forename: William E
authors.initials: WE
authors.lastname: Jack
authors.forename: Megan N
authors.initials: MN
authors.lastname: Hersh
authors.forename: Michael L
authors.initials: ML
authors.lastname: Metzker
mesh_terms: D004247:DNA / Q000096:biosynthesis* / Q000737:chemistry; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000378:metabolism*; D003856:Deoxyuracil Nucleotides / Q000737:chemistry* / Q000378:metabolism; D059014:High-Throughput Nucleotide Sequencing; D007700:Kinetics; D009578:Nitrobenzenes / Q000737:chemistry; D009711:Nucleotides / Q000737:chemistry / Q000378:metabolism; D017422:Sequence Analysis, DNA
chemical_list: D003856:Deoxyuracil Nucleotides; D009578:Nitrobenzenes; D009711:Nucleotides; D004247:DNA; D004259:DNA-Directed DNA Polymerase