Molecular design of Cy3 derivative for highly sensitive in-stem molecular beacon and its application to the wash-free FISH

Bioorg Med Chem. 2015 Apr 15;23(8):1758-62. doi: 10.1016/j.bmc.2015.02.030. Epub 2015 Feb 24.


We herein describe a novel in-stem molecular beacon (ISMB) containing multiple Cy3-quencher pairs on d-threoninol scaffolds in the stem region. The designed Cy3 derivative was not significantly quenched by the adjacent nucleobases, self-quenching of the fluorophore was minimal, and the fluorophore did not severely destabilize the duplex. Using newly designed Cy3, we synthesized ISMBs containing two Cy3 moieties. The signal to background ratio of the ISMB containing two Cy3 moieties was above 100, whereas that with one Cy3 was 30. A Cy3-derivative containing ISMB used in a fluorescence in situ hybridization (FISH) detected endogenous β-actin mRNA in fixed cells without need for washing procedures.

Keywords: Cy3; DNA; Fluorescence in situ hybridization; Molecular beacon.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Actins / genetics
  • Amino Alcohols / chemistry*
  • Base Sequence
  • Butylene Glycols / chemistry*
  • Carbocyanines / chemistry*
  • Fluorescent Dyes / chemistry*
  • HeLa Cells
  • Humans
  • In Situ Hybridization, Fluorescence / methods*
  • Oligonucleotide Probes / chemistry
  • RNA, Messenger / analysis*
  • RNA, Messenger / genetics


  • Actins
  • Amino Alcohols
  • Butylene Glycols
  • Carbocyanines
  • Fluorescent Dyes
  • Oligonucleotide Probes
  • RNA, Messenger
  • cyanine dye 3
  • threoninol