2. Academic Validation
  3. Single-Molecule Characterization of Cy3.5 -Cy5.5 Dye Pair for FRET Studies of Nucleic Acids and Nucleosomes

Single-Molecule Characterization of Cy3.5 -Cy5.5 Dye Pair for FRET Studies of Nucleic Acids and Nucleosomes

  • J Fluoresc. 2023 Mar;33(2):413-421. doi: 10.1007/s10895-022-03093-z.
Mohamed Ghoneim 1,  Catherine A Musselman 2
Affiliations

Affiliations

  • 1 Biochemistry and Molecular Genetics, University of Colorado Anschutz Medical Campus, 80045, Aurora, CO, USA. [email protected].
  • 2 Biochemistry and Molecular Genetics, University of Colorado Anschutz Medical Campus, 80045, Aurora, CO, USA.
PMID: 36435903 DOI: 10.1007/s10895-022-03093-z
Abstract

Single molecule FRET (Forster resonance energy transfer) is very powerful method for studying biomolecular binding dynamics and conformational transitions. Only a few donor - acceptor dye pairs have been characterized for use in single-molecule FRET (smFRET) studies. Hence, introducing and characterizing additional FRET dye pairs is important in order to widen the scope of applications of single-molecule FRET in biomolecular studies. Here we characterize the properties of the Cy3.5 and Cy5.5 dye pair under FRET at the single-molecule level using naked double-stranded DNA (dsDNA) and the nucleosome. We show that this pair of dyes is photostable for ~ 5 min under continuous illumination. We also report Cy3.5-Cy5.5 FRET proximity dependence and stability in the presence of several biochemical buffers and photoprotective reagents in the context of double-stranded DNA. Finally, we demonstrate compatibility of the Cy3.5-Cy5.5 pair for smFRET in vitro studies of nucleosomes.

Keywords

Cy3.5; Cy5.5; Double-stranded DNA; FRET; Nucleosome; Single-molecule fluorescence microscopy.

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