Abstract
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 fluorescence resonance energy transfer data. The positions have been demonstrated for double-stranded (ds) DNA 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 basepair such that their indole nitrogen atoms lie on the major groove side, and thus their pendant methyl groups are on the minor groove side.
Highlights
Fluorescence resonance energy transfer (FRET) has been extensively used in single-molecule studies of nucleic acid structure and dynamics [1,2,3]. This provides information on how distances between two fluorophores within DNA, RNA, or their complexes with proteins alter during structural transitions. The basis of this is the change in the efficiency of FRET (EFRET) as the interfluorophore distance changes
The exact position adopted by the fluorophores was influenced by the nature of the attachment to the DNA, and we found that the rotational setting of cyanine fluorophores altered when tethered by a longer linker [11,12]
Crystallization of cyanine fluorophoreconjugated RNA complexed with L5 protein
Summary
Fluorescence resonance energy transfer (FRET) has been extensively used in single-molecule studies of nucleic acid structure and dynamics [1,2,3]. Most commonly, this provides information on how distances between two fluorophores within DNA, RNA, or their complexes with proteins alter during structural transitions. The scalar product of these vectors is a function of the angle between them, and this complicates the extraction of absolute distance information from FRET data [4,5,6] If both fluorophores are mobile the analysis is simplified [5], but this is not always the case. The interpretation will be aided by a knowledge of how the fluorophores are oriented relative to the macromolecule to be studied
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