From LaserGen, NEB & Baylor
Rapid incorporation kinetics and improved fidelity of a novel class of 3′-OH unblocked reversible terminators
Andrew F. Gardner1, Jinchun Wang2, Weidong Wu2, Jennifer Karouby1, Hong Li2, Brian P. Stupi2, William E. Jack1, Megan N. Hersh2 and Michael L. Metzker2,3,4,*
1 New England Biolabs, Ipswich, MA 01938, 2 LaserGen, Inc., Houston, TX 77054, 3 Human Genome Sequencing Center and 4 Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA
Nucl. Acids Res. (2012) doi: 10.1093/nar/gks330
First published online: May 8, 2012
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 (kpol) that range from 35 to 45 nt/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 improved nucleotide 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.
Rapid incorporation kinetics and improved fidelity of a novel class of 3′-OH unblocked reversible terminators
Andrew F. Gardner1, Jinchun Wang2, Weidong Wu2, Jennifer Karouby1, Hong Li2, Brian P. Stupi2, William E. Jack1, Megan N. Hersh2 and Michael L. Metzker2,3,4,*
1 New England Biolabs, Ipswich, MA 01938, 2 LaserGen, Inc., Houston, TX 77054, 3 Human Genome Sequencing Center and 4 Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA
Nucl. Acids Res. (2012) doi: 10.1093/nar/gks330
First published online: May 8, 2012
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 (kpol) that range from 35 to 45 nt/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 improved nucleotide 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.