The structure of sulfoindocarbocyanine 3 terminally attached to dsDNA via a long, flexible tether.
Fluorescence resonance energy transfer (FRET) is an important source of long-range distance information in macromolecules. However, extracting maximum information requires knowledge of fluorophore, donor and acceptor, positions on the macromolecule. We previously determined the structure of the indocarbocyanine fluorophores Cy3 and Cy5 attached to DNA via three-carbon atom tethers, showing that they stacked onto the end of the helix in a manner similar to an additional basepair. Our recent FRET study has suggested that when they are attached via a longer 13-atom tether, these fluorophores are repositioned relative to the terminal basepair by a rotation of ∼30°, while remaining stacked. In this study, we have used NMR to extend our structural understanding to the commonly used fluorophore sulfoindocarbocyanine-3 (sCy3) attached to the 5'-terminus of the double-helical DNA via a 13-atom flexible tether (L13). We find that L13-sCy3 remains predominantly stacked onto the end of the duplex, but adopts a significantly different conformation, from that of either Cy3 or Cy5 attached by 3-atom tethers, with the long axes of the fluorophore and the terminal basepair approximately parallel. This result is in close agreement with our FRET data, supporting the contention that FRET data can be used to provide orientational information.
Nucleic Acid Structure Research Group, The University of Dundee, Dundee, United Kingdom.
Linas
L
Urnavicius
Scott A
SA
McPhee
David M J
DM
Lilley
David G
DG
Norman
D001483:Base Sequence; D002232:Carbocyanines / Q000737:chemistry*; D004247:DNA / Q000737:chemistry* / Q000235:genetics; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry*; D007211:Indoles / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D011522:Protons; D013451:Sulfonic Acids / Q000737:chemistry*
D002232:Carbocyanines; D005456:Fluorescent Dyes; D007211:Indoles; D011522:Protons; D013451:Sulfonic Acids; D004247:DNA