Journal
NATURE PROTOCOLS
Volume 13, Issue 4, Pages 652-665Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/nprot.2017.156
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Funding
- JSPS KAKENHI [26288083, 17H03091]
- Takeda Science Foundation
- Nakatani Foundation
- Grants-in-Aid for Scientific Research [17H03091, 26288083] Funding Source: KAKEN
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Growing evidence indicates that RNA G-quadruplexes have important roles in various processes such as transcription, translation, regulation of telomere length, and formation of telomeric heterochromatin. Investigation of RNA G-quadruplex structures associated with biological events is therefore essential to understanding the functions of these RNA molecules. We recently demonstrated that the sensitivity and simplicity of F-19 NMR can be used to directly observe higher-order telomeric G-quadruplexes of labeled RNA molecules in vitro and in living cells, as well as their interactions with ligands and proteins. This protocol describes detailed procedures for preparing F-19-labeled RNA, the evaluation of F-19-labeled RNA G-quadruplexes in vitro and in living Xenopus laevis oocytes by F-19 NMR spectroscopy, the quantitative characterization of thermodynamic properties of the G-quadruplexes, and monitoring of RNA G-quadruplex interactions with ligand molecules and proteins. This approach has several advantages over existing techniques. First, it is relatively easy to prepare F-19-labeled RNA molecules by introducing a 3,5-bis(trifluoromethyl) benzene moiety into its 5' terminus. Second, the absence of any natural fluorine background signal in RNA and cells results in a simple and clear F-19 NMR spectrum and does not suffer from high background signals as does H-1 NMR. Finally, the simplicity and sensitivity of F-19 NMR can be used to easily distinguish different RNA G-quadruplex conformations under various conditions, even in living cells, and to obtain the precise thermodynamic parameters of higher-order G-quadruplexes. This protocol can be completed in 2 weeks.
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