Bulk and single-molecule characterization of an improved molecular beacon utilizing H-dimer excitonic behavior

J Phys Chem B. 2007 Jul 19;111(28):7929-31. doi: 10.1021/jp073310d. Epub 2007 Jun 21.

Abstract

Pairs of fluorophores in close proximity often show self-quenching of fluorescence by the well-known H-dimer mechanism. We use a pair of fluorophores in the new dicyanomethylenedihydrofuran (DCDHF) dye family in the design and characterization of a new fluorescent probe for nucleic acid detection, which we refer to as a self-quenched intramolecular dimer (SQuID) molecular beacon (MB). We obtain a quenching efficiency of 97.2%, higher than the only other reported value for a MB employing fluorophore self-quenching by H-dimer formation. Furthermore, the excellent single-molecule (SM) emitter characteristics of the DCDHF dyes allow observation of individual SQuID MB-target complexes immobilized on a surface, where the doubled SM emission intensity of our target-bound beacon ensures a higher signal-to-background ratio than conventional fluorophore-quencher MBs. Additional advantages of the SQuID MB are single-pot labeling, visible colorimetric detection of the target, and intrinsic single-molecule two-step photobleaching behavior, which offers a specific means of discriminating between functional MBs and spurious fluorescence.

Publication types

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

MeSH terms

  • Base Sequence
  • Dimerization
  • Fluorescent Dyes / chemistry*
  • Furans / chemistry*
  • Molecular Sequence Data
  • Nitriles / chemistry*
  • Nucleic Acid Conformation
  • Nucleic Acid Hybridization
  • Nucleic Acids / chemistry*
  • Oligonucleotide Probes / chemistry*

Substances

  • Fluorescent Dyes
  • Furans
  • Nitriles
  • Nucleic Acids
  • Oligonucleotide Probes
  • dicyanomethylenedihydrofuran