Bioorthogonal tetrazine-mediated transfer reactions facilitate reaction turnover in nucleic acid-templated detection of microRNA

J Am Chem Soc. 2014 Dec 31;136(52):17942-5. doi: 10.1021/ja510839r. Epub 2014 Dec 19.

Abstract

Tetrazine ligations have proven to be a powerful bioorthogonal technique for the detection of many labeled biomolecules, but the ligating nature of these reactions can limit reaction turnover in templated chemistry. We have developed a transfer reaction between 7-azabenzonorbornadiene derivatives and fluorogenic tetrazines that facilitates turnover amplification of the fluorogenic response in nucleic acid-templated reactions. Fluorogenic tetrazine-mediated transfer (TMT) reaction probes can be used to detect DNA and microRNA (miRNA) templates to 0.5 and 5 pM concentrations, respectively. The endogenous oncogenic miRNA target mir-21 could be detected in crude cell lysates and detected by imaging in live cells. Remarkably, the technique is also able to differentiate between miRNA templates bearing a single mismatch with high signal to background. We imagine that TMT reactions could find wide application for amplified fluorescent detection of clinically relevant nucleic acid templates.

Publication types

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

MeSH terms

  • Aza Compounds / chemistry
  • Base Sequence
  • Biosensing Techniques / methods*
  • Fluorescent Dyes / chemistry*
  • Heterocyclic Compounds, 1-Ring / chemistry*
  • Humans
  • MCF-7 Cells
  • MicroRNAs / analysis*
  • MicroRNAs / chemistry
  • MicroRNAs / genetics
  • Models, Molecular
  • Nucleic Acid Conformation

Substances

  • Aza Compounds
  • Fluorescent Dyes
  • Heterocyclic Compounds, 1-Ring
  • MicroRNAs