DNA Primer Extension with Cyclopropenylated 7-Deaza-2'-deoxyadenosine and Efficient Bioorthogonal Labeling in Vitro and in Living Cells

Chembiochem. 2018 Sep 17;19(18):1949-1953. doi: 10.1002/cbic.201800354. Epub 2018 Aug 15.

Abstract

A deoxyadenosine triphosphate (dATP) analogue for DNA labeling was synthesized with the 1-methylcyclopropene (1MCP) group at the 7-position of 7-deaza-2'-deoxyadenosine and applied for primer extension experiments. The real-time kinetic data reveals that this 1MCP-modified dATP analogue is incorporated into DNA much faster than that of the similarly 1MCP-modified deoxyuridine triphosphate (dUTP) analogue. The postsynthetic fluorescent labeling of these oligonucleotides works efficiently according to PAGE analysis, and can be applied for immobilization of a functional antibody on a surface. Site-specific labeling at two different positions in DNA was achieved and the bioorthogonality of the postsynthetic fluorescent labeling was demonstrated in living HeLa cells.

Keywords: DNA polymerase; cycloaddition; fluorescence; imaging agents; oligonucleotides.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • Cyclopropanes / chemistry*
  • DNA / analysis*
  • DNA Primers / chemistry*
  • Deoxyadenosines / chemistry*
  • Fluorescent Dyes / chemistry*
  • HeLa Cells
  • Humans
  • Methylation
  • Microscopy, Fluorescence
  • Optical Imaging

Substances

  • Cyclopropanes
  • DNA Primers
  • Deoxyadenosines
  • Fluorescent Dyes
  • cyclopropene
  • DNA
  • 2'-deoxyadenosine