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

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.