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Probing RNA dynamics via longitudinal exchange and CPMG relaxation dispersion NMR spectroscopy using a sensitive 13C-methyl label.

Kloiber K, Spitzer R, Tollinger M, Konrat R, Kreutz C - Nucleic Acids Res. (2011)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Organic Chemistry, Leopold Franzens University, Innrain 52a, 6020 Innsbruck, Austria.

ABSTRACT
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.

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Synthesis of the 2′-O-13CH3-uridine phosphoramidite 3; (a) 6 eq. 13C-magnesium methoxide [Mg(O13CH3)2, freshly prepared from magnesium turnings and 13CH3OH] in anhydrous dimethylformamide, 100°C, 2 h, 93%; (b) 1.5 eq. (2-cyanoethyl)-N,N-diisopropylchlorophosphoramidite, 10 eq. N-ethyldimethylamine in anhydrous dichloromethane, room temperature, 2.5 h, 71%.
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S1: Synthesis of the 2′-O-13CH3-uridine phosphoramidite 3; (a) 6 eq. 13C-magnesium methoxide [Mg(O13CH3)2, freshly prepared from magnesium turnings and 13CH3OH] in anhydrous dimethylformamide, 100°C, 2 h, 93%; (b) 1.5 eq. (2-cyanoethyl)-N,N-diisopropylchlorophosphoramidite, 10 eq. N-ethyldimethylamine in anhydrous dichloromethane, room temperature, 2.5 h, 71%.


Probing RNA dynamics via longitudinal exchange and CPMG relaxation dispersion NMR spectroscopy using a sensitive 13C-methyl label.

Kloiber K, Spitzer R, Tollinger M, Konrat R, Kreutz C - Nucleic Acids Res. (2011)

Synthesis of the 2′-O-13CH3-uridine phosphoramidite 3; (a) 6 eq. 13C-magnesium methoxide [Mg(O13CH3)2, freshly prepared from magnesium turnings and 13CH3OH] in anhydrous dimethylformamide, 100°C, 2 h, 93%; (b) 1.5 eq. (2-cyanoethyl)-N,N-diisopropylchlorophosphoramidite, 10 eq. N-ethyldimethylamine in anhydrous dichloromethane, room temperature, 2.5 h, 71%.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3105391&req=5

S1: Synthesis of the 2′-O-13CH3-uridine phosphoramidite 3; (a) 6 eq. 13C-magnesium methoxide [Mg(O13CH3)2, freshly prepared from magnesium turnings and 13CH3OH] in anhydrous dimethylformamide, 100°C, 2 h, 93%; (b) 1.5 eq. (2-cyanoethyl)-N,N-diisopropylchlorophosphoramidite, 10 eq. N-ethyldimethylamine in anhydrous dichloromethane, room temperature, 2.5 h, 71%.
Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Organic Chemistry, Leopold Franzens University, Innrain 52a, 6020 Innsbruck, Austria.

ABSTRACT
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.

Show MeSH