Probing RNA dynamics via longitudinal exchange and CPMG relaxation dispersion NMR spectroscopy using a sensitive 13C-methyl label.
The refolding kinetics of bistable RNA sequences were studied in unperturbed equilibrium via (13)C exchange NMR spectroscopy. For this purpose a straightforward labeling technique was elaborated using a 2'-(13)C-methoxy uridine modification, which was prepared by a two-step synthesis and introduced into RNA using standard protocols. Using (13)C longitudinal exchange NMR spectroscopy the refolding kinetics of a 20 nt bistable RNA were characterized at temperatures between 298 and 310K, yielding the enthalpy and entropy differences between the conformers at equilibrium and the activation energy of the refolding process. The kinetics of a more stable 32 nt bistable RNA could be analyzed by the same approach at elevated temperatures, i.e. at 314 and 316 K. Finally, the dynamics of a multi-stable RNA able to fold into two hairpin- and a pseudo-knotted conformation was studied by (13)C relaxation dispersion NMR spectroscopy.
Institute of Organic Chemistry, Leopold Franzens University, Innrain 52a, 6020 Innsbruck, Austria.
Karin
K
Kloiber
Romana
R
Spitzer
Martin
M
Tollinger
Robert
R
Konrat
Christoph
C
Kreutz
D001483:Base Sequence; D002247:Carbon Isotopes; D007553:Isotope Labeling / Q000379:methods*; D007700:Kinetics; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D009690:Nucleic Acid Conformation; D009943:Organophosphorus Compounds / Q000138:chemical synthesis / Q000737:chemistry; D012313:RNA / Q000737:chemistry*; D014529:Uridine / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000737:chemistry
D002247:Carbon Isotopes; D009943:Organophosphorus Compounds; C101323:uridine phosphoramidite; D012313:RNA; D014529:Uridine
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