NMR probing of invisible excited states using selectively labeled RNAs

[nmrlearner]

NMR probing of invisible excited states using selectively labeled RNAs

Abstract

Carrâ??Purcellâ??Meiboomâ??Gill (CPMG) relaxation dispersion NMR experiments are invaluable for probing sparsely and transiently populated biomolecular states that cannot be directly detected by traditional NMR experiments and that are invisible by other biophysical approaches. A notable gap for RNA is the absence of CPMG experiments for measurement of methine base 1H and methylene C5â?² chemical shifts of ribose moieties in the excited state, partly because of complications from homonuclear 13Câ??13C scalar couplings. Here we present site-specific 13C labeling that makes possible the design of pulse sequences for recording accurate 1Hâ??13C MQ and SQ CPMG experiments for ribose methine H1â?²â??C1â?² and H2â?²â??C2â?², base and ribose 1H CPMG, as well as a new 1Hâ??13C TROSY-detected methylene (CH2) C5â?² CPMG relaxation pulse schemes. We demonstrate the utility of these experiments for two RNAs, the A-Site RNA known to undergo exchange and the IRE RNA suspected of undergoing exchange on microseconds to millisecond time-scale. We anticipate the new labeling approaches will facilitate obtaining structures of invisible states and provide insights into the relevance of such states for RNA-drug interactions.



Source: Journal of Biomolecular NMR