The Effect of dye-dye interactions on the spatial resolution of single-molecule FRET measurements in nucleic acids.
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 approximately 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.
Di Fiori
Nicolas
N
Department of Physics, Boston University, Boston, Massachusetts, USA.
Meller
Amit
A
D002232:Carbocyanines / Q000737:chemistry; D004396:Coloring Agents / Q000737:chemistry*; D004247:DNA / Q000032:analysis; D019281:Dimerization; D005453:Fluorescence; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes; D008163:Luminescent Measurements; D008956:Models, Chemical; D009690:Nucleic Acid Conformation / Q000187:drug effects*; D009696:Nucleic Acids / Q000032:analysis* / Q000737:chemistry; D010777:Photochemistry; D013050:Spectrometry, Fluorescence / Q000379:methods
D002232:Carbocyanines; D004396:Coloring Agents; D005456:Fluorescent Dyes; D009696:Nucleic Acids; C085321:cyanine dye 5; D004247:DNA