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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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Analysis of CPMG relaxation dispersion of RNA sequence 6. A two-state process that is fast on the chemical shift time-scale was fit to the data of conformer 6′ (A) and 6′′′ (B) (for details of the fitting procedure see text and Supplementary Information). Experimental data points are shown as black circles and the fit is depicted as a red line. Spectrometer field strengths are indicated and the secondary structures of the two states are shown as inserts. Both conformers 6′ and 6′′′ exchange with their respective excited state at a rate constant of ∼500 s−1.
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Figure 7: Analysis of CPMG relaxation dispersion of RNA sequence 6. A two-state process that is fast on the chemical shift time-scale was fit to the data of conformer 6′ (A) and 6′′′ (B) (for details of the fitting procedure see text and Supplementary Information). Experimental data points are shown as black circles and the fit is depicted as a red line. Spectrometer field strengths are indicated and the secondary structures of the two states are shown as inserts. Both conformers 6′ and 6′′′ exchange with their respective excited state at a rate constant of ∼500 s−1.

Mentions: We recorded experiments at 300 K at 125 and 200 MHz carbon frequency. Small but statistically significant dispersion profiles could be found for conformers 6′ and 6′′′ (the least populated conformer 6′′ could not be analyzed due to low signal to noise ratio, Figure 7). In order to assess whether the underlying process is the slow exchange between the visible conformers, we analyzed the data using equations for two-state processes on the very slow, the intermediate and the fast time regimes (34,35). Also slow exchange in 2 three-state exchange models was investigated (Supplementary Table S5).Figure 7.


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)

Analysis of CPMG relaxation dispersion of RNA sequence 6. A two-state process that is fast on the chemical shift time-scale was fit to the data of conformer 6′ (A) and 6′′′ (B) (for details of the fitting procedure see text and Supplementary Information). Experimental data points are shown as black circles and the fit is depicted as a red line. Spectrometer field strengths are indicated and the secondary structures of the two states are shown as inserts. Both conformers 6′ and 6′′′ exchange with their respective excited state at a rate constant of ∼500 s−1.
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Related In: Results  -  Collection

License
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Figure 7: Analysis of CPMG relaxation dispersion of RNA sequence 6. A two-state process that is fast on the chemical shift time-scale was fit to the data of conformer 6′ (A) and 6′′′ (B) (for details of the fitting procedure see text and Supplementary Information). Experimental data points are shown as black circles and the fit is depicted as a red line. Spectrometer field strengths are indicated and the secondary structures of the two states are shown as inserts. Both conformers 6′ and 6′′′ exchange with their respective excited state at a rate constant of ∼500 s−1.
Mentions: We recorded experiments at 300 K at 125 and 200 MHz carbon frequency. Small but statistically significant dispersion profiles could be found for conformers 6′ and 6′′′ (the least populated conformer 6′′ could not be analyzed due to low signal to noise ratio, Figure 7). In order to assess whether the underlying process is the slow exchange between the visible conformers, we analyzed the data using equations for two-state processes on the very slow, the intermediate and the fast time regimes (34,35). Also slow exchange in 2 three-state exchange models was investigated (Supplementary Table S5).Figure 7.

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