Crystal structures of cyanine fluorophores stacked onto the end of double-stranded RNA

Yijin Liu, David M. J. Lilley (Lead / Corresponding author)

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    8 Citations (Scopus)
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    The indodicarbocyanine fluorophores Cy3 and Cy5 are extensively used as donor-acceptor pairs in fluorescence resonance energy transfer experiments, especially those involving single molecules. When terminally-attached to double-stranded nucleic acids via the 5' phosphate group these fluorophores stack onto the ends of the molecule. Knowledge of the positions of the fluorophores is critical to the interpretation of FRET data. The positions have been demonstrated for dsDNA using NMR spectroscopy. Here we have used X-ray crystallography to analyze the location of Cy3 and Cy5 on dsRNA, using complexes of an RNA stem-loop bound to L5 protein determined at 2.4 Å resolution. This confirms the tendency of both fluorophores to stack on the free end of RNA, with the long axis of the fluorophores approximately parallel to that of the terminal basepair. However, the manner of interaction of both Cy3 and Cy5 with the terminus of the dsRNA is significantly different from that deduced for dsDNA using NMR. The fluorophores are stacked on the terminal base pair such that their indole nitrogen atoms lie on the major groove side, and thus their pendant methyl groups are on the minor groove side.
    Original languageEnglish
    Pages (from-to)2236-2343
    Number of pages8
    JournalBiophysical Journal
    Issue number11
    Publication statusPublished - 5 Dec 2017


    • Cy3
    • Cy5 structure
    • FRET
    • Fluorescence
    • X-ray diffraction
    • RNA-protein interaction


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