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

Nucleic Acids Res. 2012 Aug;40(15):7404-15. doi: 10.1093/nar/gks330. Epub 2012 May 8.


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.

MeSH terms

  • DNA / biosynthesis*
  • DNA / chemistry
  • DNA-Directed DNA Polymerase / chemistry
  • DNA-Directed DNA Polymerase / metabolism*
  • Deoxyuracil Nucleotides / chemistry*
  • Deoxyuracil Nucleotides / metabolism
  • High-Throughput Nucleotide Sequencing
  • Kinetics
  • Nitrobenzenes / chemistry
  • Nucleotides / chemistry
  • Nucleotides / metabolism
  • Sequence Analysis, DNA


  • Deoxyuracil Nucleotides
  • Nitrobenzenes
  • Nucleotides
  • DNA
  • DNA-Directed DNA Polymerase