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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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Kinetics of sequence 4 analyzed by 13C longitudinal exchange spectroscopy. (A) Interconversion between conformations 4′ and 4′′. The uridine nucleotide that serves as a sensor is highlighted in red. (B) Signal intensities as a function of mixing time are shown for a set of temperatures. The left panel shows normalized intensities of the correlation peak of conformation 4′ and the exchange peak corresponding to transition 4′ → 4′′. The right panel depicts the intensities of the correlation peak of conformation 4′′ and the exchange peak corresponding to transition 4′′ → 4′. Experiments performed at different temperatures are color-coded (298 K: blue, 300 K: magenta, 303 K: red, 306 K: orange, 310 K: yellow).
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Figure 4: Kinetics of sequence 4 analyzed by 13C longitudinal exchange spectroscopy. (A) Interconversion between conformations 4′ and 4′′. The uridine nucleotide that serves as a sensor is highlighted in red. (B) Signal intensities as a function of mixing time are shown for a set of temperatures. The left panel shows normalized intensities of the correlation peak of conformation 4′ and the exchange peak corresponding to transition 4′ → 4′′. The right panel depicts the intensities of the correlation peak of conformation 4′′ and the exchange peak corresponding to transition 4′′ → 4′. Experiments performed at different temperatures are color-coded (298 K: blue, 300 K: magenta, 303 K: red, 306 K: orange, 310 K: yellow).

Mentions: We recorded a 13C, 1H resolved longitudinal exchange experiment which yields a series of 13C, 1H correlation maps with correlation- and exchange-peaks that have been amplitude modulated during a mixing time according to their longitudinal relaxation rates and their kinetics of interconversion. Decays and buildups at the different temperatures are shown in Figure 4. Assuming a two-state process we determined the refolding kinetics in the temperature range between 298 and 310 K yielding microscopic exchange rate constants k4′→4′′ between 0.05 and 0.42 s−1 and k4′′→4′ between 0.04 and 0.16 s−1, with errors being on the order of 5–10% (Table 1). This corresponds to an exchange rate constant kex (kex = k4′→4′′ + k4′′→4′) between 0.09 and 0.58 s−1 and the population distribution of p4′/p4′′ changing from 0.57/0.43 to 0.73/0.27, which agrees well with the values obtained from integration of peak volumes (0.50/0.50 to 0.65/0.35 in the corresponding temperature range).Figure 4.


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)

Kinetics of sequence 4 analyzed by 13C longitudinal exchange spectroscopy. (A) Interconversion between conformations 4′ and 4′′. The uridine nucleotide that serves as a sensor is highlighted in red. (B) Signal intensities as a function of mixing time are shown for a set of temperatures. The left panel shows normalized intensities of the correlation peak of conformation 4′ and the exchange peak corresponding to transition 4′ → 4′′. The right panel depicts the intensities of the correlation peak of conformation 4′′ and the exchange peak corresponding to transition 4′′ → 4′. Experiments performed at different temperatures are color-coded (298 K: blue, 300 K: magenta, 303 K: red, 306 K: orange, 310 K: yellow).
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Figure 4: Kinetics of sequence 4 analyzed by 13C longitudinal exchange spectroscopy. (A) Interconversion between conformations 4′ and 4′′. The uridine nucleotide that serves as a sensor is highlighted in red. (B) Signal intensities as a function of mixing time are shown for a set of temperatures. The left panel shows normalized intensities of the correlation peak of conformation 4′ and the exchange peak corresponding to transition 4′ → 4′′. The right panel depicts the intensities of the correlation peak of conformation 4′′ and the exchange peak corresponding to transition 4′′ → 4′. Experiments performed at different temperatures are color-coded (298 K: blue, 300 K: magenta, 303 K: red, 306 K: orange, 310 K: yellow).
Mentions: We recorded a 13C, 1H resolved longitudinal exchange experiment which yields a series of 13C, 1H correlation maps with correlation- and exchange-peaks that have been amplitude modulated during a mixing time according to their longitudinal relaxation rates and their kinetics of interconversion. Decays and buildups at the different temperatures are shown in Figure 4. Assuming a two-state process we determined the refolding kinetics in the temperature range between 298 and 310 K yielding microscopic exchange rate constants k4′→4′′ between 0.05 and 0.42 s−1 and k4′′→4′ between 0.04 and 0.16 s−1, with errors being on the order of 5–10% (Table 1). This corresponds to an exchange rate constant kex (kex = k4′→4′′ + k4′′→4′) between 0.09 and 0.58 s−1 and the population distribution of p4′/p4′′ changing from 0.57/0.43 to 0.73/0.27, which agrees well with the values obtained from integration of peak volumes (0.50/0.50 to 0.65/0.35 in the corresponding temperature range).Figure 4.

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