Abstract
We study the effect of dye-dye interactions in labeled double-stranded DNA molecules on the Förster resonance energy transfer (FRET) efficiency at the single-molecule level. An extensive analysis of internally labeled double-stranded DNA molecules in bulk and at the single-molecule level reveals that donor-acceptor absolute distances can be reliably extracted down to ∼3-nm separation, provided that dye-dye quenching is accounted for. At these short separations, we find significant long-lived fluorescence fluctuations among discrete levels originating from the simultaneous and synchronous quenching of both dyes. By comparing four different donor-acceptor dye pairs (TMR-ATTO647N, Cy3-ATTO647N, TMR-Cy5, and Cy3-Cy5), we find that this phenomenon depends on the nature of the dye pair used, with the cyanine pair Cy3-Cy5 showing the least amount of fluctuations. The significance of these results is twofold: First, they illustrate that when dye-dye quenching is accounted for, single-molecule FRET can be used to accurately measure inter-dye distances, even at short separations. Second, these results are useful when deciding which dye pairs to use for nucleic acids analyses using FRET.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.