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 macromo...

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Bibliographic Details
Published in:Biochemistry (Easton) 2008-07, Vol.47 (30), p.7857-7862
Main Authors: Iqbal, Asif, Wang, Lihua, Thompson, Katherine C, Lilley, David M. J, Norman, David G
Format: Article
Language:English
Subjects:
Base Sequence
Carbocyanines - chemistry
Carbocyanines - metabolism
DNA - chemistry
DNA - metabolism
Fluorescence Resonance Energy Transfer - methods
Magnetic Resonance Spectroscopy
Molecular Sequence Data
Molecular Structure
Nucleic Acid Conformation
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Summary: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.
ISSN:0006-2960
1520-4995
DOI:10.1021/bi800773f