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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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 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
Volume113
Issue number11
DOIs
Publication statusPublished - 5 Dec 2017

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Double-Stranded RNA
RNA
Fluorescence Resonance Energy Transfer
X Ray Crystallography
Base Pairing
Nucleic Acids
Magnetic Resonance Spectroscopy
Nitrogen
Phosphates
cyanine dye 5
Proteins

Keywords

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

Cite this

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title = "Crystal structures of cyanine fluorophores stacked onto the end of double-stranded RNA",
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 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 {\AA} 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.",
keywords = "Cy3, Cy5 structure, FRET, Fluorescence, X-ray diffraction, RNA-protein interaction",
author = "Yijin Liu and Lilley, {David M. J.}",
note = "The authors thank Scott McPhee and Saira Ashraf for chemical synthesis of fluorophore-labeled RNA and Drs. Tim Wilson and David Norman for discussion. Cancer Research-UK provided financial support via program grant A18604, and the Wellcome Trust funded the in-house diffractometer. ESRF provided synchrotron beam time.",
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T1 - Crystal structures of cyanine fluorophores stacked onto the end of double-stranded RNA

AU - Liu, Yijin

AU - Lilley, David M. J.

N1 - The authors thank Scott McPhee and Saira Ashraf for chemical synthesis of fluorophore-labeled RNA and Drs. Tim Wilson and David Norman for discussion. Cancer Research-UK provided financial support via program grant A18604, and the Wellcome Trust funded the in-house diffractometer. ESRF provided synchrotron beam time.

PY - 2017/12/5

Y1 - 2017/12/5

N2 - 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.

AB - 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.

KW - Cy3

KW - Cy5 structure

KW - FRET

KW - Fluorescence

KW - X-ray diffraction

KW - RNA-protein interaction

U2 - 10.1016/j.bpj.2017.10.002

DO - 10.1016/j.bpj.2017.10.002

M3 - Article

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JO - Biophysical Journal

JF - Biophysical Journal

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