Pyrene-based quantitative detection of the 5-formylcytosine loci symmetry in the CpG duplex content during TET-dependent demethylation

Angew Chem Int Ed Engl. 2014 Oct 13;53(42):11223-7. doi: 10.1002/anie.201406220. Epub 2014 Aug 27.

Abstract

Methylcytosine (5mC) is mostly symmetrically distributed in CpG sites. Ten-eleven-translocation (TET) proteins are the key enzymes involved in active DNA demethylation through stepwise oxidation of 5mC. However, oxidation pathways of TET enzymes in the symmetrically methylated CpG context are still elusive. Employing the unique fluorescence properties of pyrene group, we designed and synthesized a sensitive fluorescence-based probe not only to target 5-formylcytosine (5fC) sites, but also to distinguish symmetric from asymmetric 5fC sites in the double stranded DNA context during TET-dependent 5mC oxidation process. Using this novel probe, we revealed dominant levels of symmetric 5fC among total 5fC sites during in vitro TET-dependent 5mC oxidation and novel mechanistic insights into the TET-dependent 5mC oxidation in the mCpG context.

Keywords: 5-formylcytosine; DNA demethylation; TET oxidation; pyrene fluorescence.

Publication types

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

MeSH terms

  • CpG Islands*
  • Cytosine / analogs & derivatives*
  • Cytosine / analysis
  • DNA / chemistry*
  • DNA / metabolism
  • DNA Methylation*
  • DNA-Binding Proteins / metabolism
  • Fluorescent Dyes / analysis*
  • Fluorescent Dyes / chemical synthesis
  • Oxidation-Reduction
  • Pyrenes / analysis*
  • Pyrenes / chemical synthesis
  • Signal Transduction
  • Spectrometry, Fluorescence

Substances

  • 5-formylcytosine
  • DNA-Binding Proteins
  • Fluorescent Dyes
  • Pyrenes
  • Cytosine
  • DNA
  • pyrene