Design and synthesis of a 3'-O-allyl photocleavable fluorescent nucleotide as a reversible terminator for DNA sequencing by synthesis

Proc Natl Acad Sci U S A. 2005 Apr 26;102(17):5932-7. doi: 10.1073/pnas.0501962102. Epub 2005 Apr 13.


DNA sequencing by synthesis (SBS) offers an approach for potential high-throughput sequencing applications. In this method, the ability of an incoming nucleotide to act as a reversible terminator for a DNA polymerase reaction is an important requirement to unambiguously determine the identity of the incorporated nucleotide before the next nucleotide is added. A free 3'-OH group on the terminal nucleotide of the primer is necessary for the DNA polymerase to incorporate an incoming nucleotide. Therefore, if the 3'-OH group of an incoming nucleotide is capped by a chemical moiety, it will cause the polymerase reaction to terminate after the nucleotide is incorporated into the DNA strand. If the capping group is subsequently removed to generate a free 3'-OH, the polymerase reaction will reinitialize. We report here the design and synthesis of a 3'-modified photocleavable fluorescent nucleotide, 3'-O-allyl-dUTP-PC-Bodipy-FL-510 (PC-Bodipy, photocleavable 4,4-difluoro-4-bora-3alpha,4alpha-diaza-s-indacene), as a reversible terminator for SBS. This nucleotide analogue contains an allyl moiety capping the 3'-OH group and a fluorophore Bodipy-FL-510 linked to the 5 position of the uracil through a photocleavable 2-nitrobenzyl linker. Here, we have shown that this nucleotide is a good substrate for a DNA polymerase. After the nucleotide was successfully incorporated into a growing DNA strand and the fluorophore was photocleaved, the allyl group was removed by using a Pd-catalyzed reaction to reinitiate the polymerase reaction, thereby establishing the feasibility of using such nucleotide analogues as reversible terminators for SBS.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Base Sequence
  • Boron Compounds
  • DNA / chemical synthesis*
  • DNA / chemistry*
  • DNA Primers / chemical synthesis
  • DNA Primers / chemistry
  • Deoxyribonucleotides*
  • Fluorescent Dyes*
  • Indicators and Reagents
  • Nucleic Acid Conformation
  • Oligodeoxyribonucleotides / chemical synthesis
  • Oligodeoxyribonucleotides / chemistry
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization


  • 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene
  • Boron Compounds
  • DNA Primers
  • Deoxyribonucleotides
  • Fluorescent Dyes
  • Indicators and Reagents
  • Oligodeoxyribonucleotides
  • DNA