Orientation dependence in fluorescent energy transfer between Cy3 and Cy5 terminally attached to double-stranded nucleic acids

Asif Iqbal, Sinan Arslan, Burak Okumus, Timothy J. Wilson, Gerard Giraud, David G. Norman, Taekjip Ha, David M.J. Lilley

Research output: Contribution to journalArticlepeer-review

Abstract

We have found that the efficiency of fluorescence resonance energy transfer between Cy3 and Cy5 terminally attached to the 5′ ends of a DNA duplex is significantly affected by the relative orientation of the two fluorophores. The cyanine fluorophores are predominantly stacked on the ends of the helix in the manner of an additional base pair, and thus their relative orientation depends on the length of the helix. Observed fluorescence resonance energy transfer (FRET) efficiency depends on the length of the helix, as well as its helical periodicity. By changing the helical geometry from B form double-stranded DNA to A form hybrid RNA/DNA, a marked phase shift occurs in the modulation of FRET efficiency with helix length. Both curves are well explained by the standard geometry of B and A form helices. The observed modulation for both polymers is less than that calculated for a fully rigid attachment of the fluorophores. However, a model involving lateral mobility of the fluorophores on the ends of the helix explains the observed experimental data. This has been further modified to take account of a minor fraction of unstacked fluorophore observed by fluorescent lifetime measurements. Our data unequivocally establish that Förster transfer obeys the orientation dependence as expected for a dipole-dipole interaction.

Original languageEnglish (US)
Pages (from-to)11176-11181
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume105
Issue number32
DOIs
StatePublished - Aug 12 2008
Externally publishedYes

Keywords

  • Cyanine fluorophores
  • FRET
  • Kappa squared
  • Single-molecule FRET

ASJC Scopus subject areas

  • General

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