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 19F 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 19F-labeled RNA, the 
evaluation of 19F-labeled RNA G-quadruplexes in vitro and in living Xenopus 
laevis oocytes by 19F 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 19F-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 19F NMR 
spectrum and does not suffer from high background signals as does 1H NMR. 
Finally, the simplicity and sensitivity of 19F 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.