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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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13C longitudinal exchange experiments conducted on sequence 5. (A) Interconversion between conformations 5′ and 5′′ with the uridine label highlighted in red. (B) Left panel: intensities of the corrrelation peak corresponding to conformation 5′ and exchange peak corresponding to transition 5′ → 5′′ as a function of mixing time. Right panel: correlation peak pertinent to fold 5′′ and exchange peak for the transition 5′′ → 5′. Results at 314 (316) K are depicted as circles (diamonds) and fits at 314 (316) K are shown as solid/dashed lines. Error bars were obtained on the basis of spectral noise in a Monte Carlo analysis.
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Figure 6: 13C longitudinal exchange experiments conducted on sequence 5. (A) Interconversion between conformations 5′ and 5′′ with the uridine label highlighted in red. (B) Left panel: intensities of the corrrelation peak corresponding to conformation 5′ and exchange peak corresponding to transition 5′ → 5′′ as a function of mixing time. Right panel: correlation peak pertinent to fold 5′′ and exchange peak for the transition 5′′ → 5′. Results at 314 (316) K are depicted as circles (diamonds) and fits at 314 (316) K are shown as solid/dashed lines. Error bars were obtained on the basis of spectral noise in a Monte Carlo analysis.

Mentions: We subsequently applied the longitudinal exchange approach to the thermodynamically more stable 32 nt RNA stem loop structure 5 that coexists in two distinct conformations 5′ and 5′′. In order to ensure that the reaction was fast enough to be observable by this approach, experiments were conducted at elevated temperatures. For this RNA it was possible to obtain meaningful results from experiments conducted at 314 and 316 K. Respective correlation- and exchange-peak intensities as a function of mixing time are shown in Figure 6. The rate of interconversion at this temperature is in the same range as for sequence 4 at room temperature, i.e. k5′→5′′ = 0.348 ± 0.063 s−1 (0.323 ± 0.056 s−1) and k5′′→5′ = 0.206 ± 0.024 s−1 (0.262 ± 0.024 s−1) at 314 K (316 K). The complete set of kinetic parameters is given in Table 2. Due to the more pronounced skew in populations (approximately 7/3) as compared to RNA sequence 4, the exchange peak derived from the less populated species displays relatively low intensities in the longitudinal exchange experiments, which, in conjunction with a small rate of interconversion, impeded the acquisition of data of satisfactory signal-to-noise ratio below 314 K. On the other hand, rapid sample degradation and thermal denaturing effects did not permit extended exposure of this RNA to higher temperatures. The temperature range investigated for sequence 5 was thus too narrow to allow reliable determination of thermodynamic and kinetic information. For even more stable RNA structures and/or more skewed populations it is advisable to resort to real-time NMR techniques (14).Figure 6.


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)

13C longitudinal exchange experiments conducted on sequence 5. (A) Interconversion between conformations 5′ and 5′′ with the uridine label highlighted in red. (B) Left panel: intensities of the corrrelation peak corresponding to conformation 5′ and exchange peak corresponding to transition 5′ → 5′′ as a function of mixing time. Right panel: correlation peak pertinent to fold 5′′ and exchange peak for the transition 5′′ → 5′. Results at 314 (316) K are depicted as circles (diamonds) and fits at 314 (316) K are shown as solid/dashed lines. Error bars were obtained on the basis of spectral noise in a Monte Carlo analysis.
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Figure 6: 13C longitudinal exchange experiments conducted on sequence 5. (A) Interconversion between conformations 5′ and 5′′ with the uridine label highlighted in red. (B) Left panel: intensities of the corrrelation peak corresponding to conformation 5′ and exchange peak corresponding to transition 5′ → 5′′ as a function of mixing time. Right panel: correlation peak pertinent to fold 5′′ and exchange peak for the transition 5′′ → 5′. Results at 314 (316) K are depicted as circles (diamonds) and fits at 314 (316) K are shown as solid/dashed lines. Error bars were obtained on the basis of spectral noise in a Monte Carlo analysis.
Mentions: We subsequently applied the longitudinal exchange approach to the thermodynamically more stable 32 nt RNA stem loop structure 5 that coexists in two distinct conformations 5′ and 5′′. In order to ensure that the reaction was fast enough to be observable by this approach, experiments were conducted at elevated temperatures. For this RNA it was possible to obtain meaningful results from experiments conducted at 314 and 316 K. Respective correlation- and exchange-peak intensities as a function of mixing time are shown in Figure 6. The rate of interconversion at this temperature is in the same range as for sequence 4 at room temperature, i.e. k5′→5′′ = 0.348 ± 0.063 s−1 (0.323 ± 0.056 s−1) and k5′′→5′ = 0.206 ± 0.024 s−1 (0.262 ± 0.024 s−1) at 314 K (316 K). The complete set of kinetic parameters is given in Table 2. Due to the more pronounced skew in populations (approximately 7/3) as compared to RNA sequence 4, the exchange peak derived from the less populated species displays relatively low intensities in the longitudinal exchange experiments, which, in conjunction with a small rate of interconversion, impeded the acquisition of data of satisfactory signal-to-noise ratio below 314 K. On the other hand, rapid sample degradation and thermal denaturing effects did not permit extended exposure of this RNA to higher temperatures. The temperature range investigated for sequence 5 was thus too narrow to allow reliable determination of thermodynamic and kinetic information. For even more stable RNA structures and/or more skewed populations it is advisable to resort to real-time NMR techniques (14).Figure 6.

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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