The Structure of Sulfoindocarbocyanine 3 Terminally Attached to dsDNA via a Long, Flexible Tether

Linas Urnavicius, Scott A. McPhee, David M. J. Lilley, David G. Norman

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    19 Citations (Scopus)

    Abstract

    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 base-pair. 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 similar to 30 degrees, 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.

    Original languageEnglish
    Pages (from-to)561-568
    Number of pages8
    JournalBiophysical Journal
    Volume102
    Issue number3
    DOIs
    Publication statusPublished - 8 Feb 2012

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