The structure of cyanine 5 terminally attached to double-stranded DNA: implications for FRET studies.
Fluorescence resonance energy transfer, FRET, can be used to obtain long-range distance information for macromolecules and is particularly powerful when used in single-molecule studies. The determination of accurate distances requires knowledge of the fluorophore position with respect to the macromolecule. In this study we have used NMR to determine the structure of the commonly used fluorophore indocarbocyanine-5 (Cy5) covalently attached to the 5'-terminus of double-helical DNA. We find that Cy5 is predominantly stacked onto the end of the duplex, in a manner similar to an additional base pair. This is very similar to the behavior of Cy3 terminally attached to DNA and suggests that the efficiency of energy transfer between Cy3 and Cy5, that are attached to nucleic acids in this way, will exhibit significant dependence on fluorophore orientation.
Iqbal
Asif
A
Cancer Research UK Nucleic Acid Structure Research Group, MSI/WTB Complex, The University of Dundee, UK.
Wang
Lihua
L
Thompson
Katherine C
KC
Lilley
David M J
DM
Norman
David G
DG
D001483:Base Sequence; D002232:Carbocyanines / Q000737:chemistry* / Q000378:metabolism; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009690:Nucleic Acid Conformation
D002232:Carbocyanines; C085321:cyanine dye 5; D004247:DNA