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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 the refolding reaction of sequence 4 in terms of kinetic and thermodynamic parameters obtained from longitudinal exchange experiments on the 2′-O-13CH3-uridine label. (A) ln (k4′→4′′) (solid line) and ln (k4′′→4′) (dashed line) as a function of the inverse temperature. (B) ln (K) [computed as ln (k4′→4′′/k4′′→4′)] as a function of 1/T. Error bars were obtained on the basis of duplicate data points by a Monte Carlo analysis, and regression was performed on averaged values of k4′→4′′, k4′′→4′, and K.
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Figure 5: Analysis of the refolding reaction of sequence 4 in terms of kinetic and thermodynamic parameters obtained from longitudinal exchange experiments on the 2′-O-13CH3-uridine label. (A) ln (k4′→4′′) (solid line) and ln (k4′′→4′) (dashed line) as a function of the inverse temperature. (B) ln (K) [computed as ln (k4′→4′′/k4′′→4′)] as a function of 1/T. Error bars were obtained on the basis of duplicate data points by a Monte Carlo analysis, and regression was performed on averaged values of k4′→4′′, k4′′→4′, and K.

Mentions: Analysis of the temperature dependence of the equilibrium constant obtained from the microscopic exchange rates yielded a refolding enthalpy and entropy of ΔH4′′-4′ = 11.2 ± 1.1 kcal mol−1 and ΔS4′′-4′ = 38.1 ± 3.7 cal mol−1 K−1. The temperature dependence of the equilibrium constant and the microscopic rate constants are shown in Figure 5. Thus, the results from the analysis of longitudinal exchange experiments are in line with those directly obtained from peak integration (ΔH4′′-4′ = 9.9 kcal mol−1, ΔS4′′-4′ = 33.1 cal mol−1 K−1). Assuming an Arrhenius temperature dependence of the rate constants, activation energies and approximated frequency factors EA4#-4′ = 32.8 ± 0.68 kcal mol−1/A4#-4′ = 1023.1 and EA4#-4′′ = 21.6 ± 0.88 kcal mol−1/A4#-4′′ = 1015.0 were obtained. The activation energy was found to amount to 55% (42%) of the (base pairing and stacking) enthalpy as computed by m-fold for fold 4′ (4′′) (44). This makes a dissociative mechanism, which would imply a fully unfolded state, rather unlikely, but favors an associative mechanism with base pairs of both conformational states formed and being disrupted at the same time during the transition state (13).Figure 5.


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 the refolding reaction of sequence 4 in terms of kinetic and thermodynamic parameters obtained from longitudinal exchange experiments on the 2′-O-13CH3-uridine label. (A) ln (k4′→4′′) (solid line) and ln (k4′′→4′) (dashed line) as a function of the inverse temperature. (B) ln (K) [computed as ln (k4′→4′′/k4′′→4′)] as a function of 1/T. Error bars were obtained on the basis of duplicate data points by a Monte Carlo analysis, and regression was performed on averaged values of k4′→4′′, k4′′→4′, and K.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
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Figure 5: Analysis of the refolding reaction of sequence 4 in terms of kinetic and thermodynamic parameters obtained from longitudinal exchange experiments on the 2′-O-13CH3-uridine label. (A) ln (k4′→4′′) (solid line) and ln (k4′′→4′) (dashed line) as a function of the inverse temperature. (B) ln (K) [computed as ln (k4′→4′′/k4′′→4′)] as a function of 1/T. Error bars were obtained on the basis of duplicate data points by a Monte Carlo analysis, and regression was performed on averaged values of k4′→4′′, k4′′→4′, and K.
Mentions: Analysis of the temperature dependence of the equilibrium constant obtained from the microscopic exchange rates yielded a refolding enthalpy and entropy of ΔH4′′-4′ = 11.2 ± 1.1 kcal mol−1 and ΔS4′′-4′ = 38.1 ± 3.7 cal mol−1 K−1. The temperature dependence of the equilibrium constant and the microscopic rate constants are shown in Figure 5. Thus, the results from the analysis of longitudinal exchange experiments are in line with those directly obtained from peak integration (ΔH4′′-4′ = 9.9 kcal mol−1, ΔS4′′-4′ = 33.1 cal mol−1 K−1). Assuming an Arrhenius temperature dependence of the rate constants, activation energies and approximated frequency factors EA4#-4′ = 32.8 ± 0.68 kcal mol−1/A4#-4′ = 1023.1 and EA4#-4′′ = 21.6 ± 0.88 kcal mol−1/A4#-4′′ = 1015.0 were obtained. The activation energy was found to amount to 55% (42%) of the (base pairing and stacking) enthalpy as computed by m-fold for fold 4′ (4′′) (44). This makes a dissociative mechanism, which would imply a fully unfolded state, rather unlikely, but favors an associative mechanism with base pairs of both conformational states formed and being disrupted at the same time during the transition state (13).Figure 5.

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
Related in: MedlinePlus