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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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Temperature dependence of folding state populations of RNA sequence 6. (A) Representation of the three possible folding states. The red U denotes the 2′-O-13CH3-uridine label. (B) 1H, 13C-HSQC spectra of RNA sequence 6 at various temperatures ranging from 278 to 303 K. (C) Folding state populations (in %) versus temperature. The populations were determined as the mean value from three independent HSQC measurements at the indicated temperature (error bars from standard deviation). Conditions: 0.75 mM RNA, 2.5 mM MgCl2, 50 mM sodium phosphate, pH 6.5, H2O/D2O 9/1, 298 K.
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Figure 3: Temperature dependence of folding state populations of RNA sequence 6. (A) Representation of the three possible folding states. The red U denotes the 2′-O-13CH3-uridine label. (B) 1H, 13C-HSQC spectra of RNA sequence 6 at various temperatures ranging from 278 to 303 K. (C) Folding state populations (in %) versus temperature. The populations were determined as the mean value from three independent HSQC measurements at the indicated temperature (error bars from standard deviation). Conditions: 0.75 mM RNA, 2.5 mM MgCl2, 50 mM sodium phosphate, pH 6.5, H2O/D2O 9/1, 298 K.

Mentions: The coexistence of the three folding states was revealed by the presence of three observable resonances in the 1H, 13C correlation map of RNA 6 (Figure 2C). By incorporation of the 2′-O-13C-methyl uridine label into sequence 6 at position 16 and into the respective reference sequences (6a and 6b), it was possible to assign the methyl 1H/13C resonances. Peak integration of the 1H, 13C-HSQC correlation maps yields an estimate of the individual populations, namely a ratio of ∼4/1/2 of pseudoknot 6′, 5′-hairpin 6′′ and 3′-hairpin 6′′′ at 298 K. The population distribution was found to be strongly temperature dependent according to peak volumes obtained at various temperatures (278, 283, 298 and 303 K, Figure 3B). While the pseudoknotted conformation 6′ was favored at lower temperatures, the hairpin folds 6′′ and 6′′′ were increasingly populated at higher temperatures (Figure 3C). This strong temperature dependence is indicative of enthalpy–entropy compensation determining the equilibrium distributions of the three species. Whereas the pseudoknotted conformation is enthalpically favored (8 bp), it is entropically destabilized with respect to the hairpin structures that have long unstructured regions.Figure 3.


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)

Temperature dependence of folding state populations of RNA sequence 6. (A) Representation of the three possible folding states. The red U denotes the 2′-O-13CH3-uridine label. (B) 1H, 13C-HSQC spectra of RNA sequence 6 at various temperatures ranging from 278 to 303 K. (C) Folding state populations (in %) versus temperature. The populations were determined as the mean value from three independent HSQC measurements at the indicated temperature (error bars from standard deviation). Conditions: 0.75 mM RNA, 2.5 mM MgCl2, 50 mM sodium phosphate, pH 6.5, H2O/D2O 9/1, 298 K.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 3: Temperature dependence of folding state populations of RNA sequence 6. (A) Representation of the three possible folding states. The red U denotes the 2′-O-13CH3-uridine label. (B) 1H, 13C-HSQC spectra of RNA sequence 6 at various temperatures ranging from 278 to 303 K. (C) Folding state populations (in %) versus temperature. The populations were determined as the mean value from three independent HSQC measurements at the indicated temperature (error bars from standard deviation). Conditions: 0.75 mM RNA, 2.5 mM MgCl2, 50 mM sodium phosphate, pH 6.5, H2O/D2O 9/1, 298 K.
Mentions: The coexistence of the three folding states was revealed by the presence of three observable resonances in the 1H, 13C correlation map of RNA 6 (Figure 2C). By incorporation of the 2′-O-13C-methyl uridine label into sequence 6 at position 16 and into the respective reference sequences (6a and 6b), it was possible to assign the methyl 1H/13C resonances. Peak integration of the 1H, 13C-HSQC correlation maps yields an estimate of the individual populations, namely a ratio of ∼4/1/2 of pseudoknot 6′, 5′-hairpin 6′′ and 3′-hairpin 6′′′ at 298 K. The population distribution was found to be strongly temperature dependent according to peak volumes obtained at various temperatures (278, 283, 298 and 303 K, Figure 3B). While the pseudoknotted conformation 6′ was favored at lower temperatures, the hairpin folds 6′′ and 6′′′ were increasingly populated at higher temperatures (Figure 3C). This strong temperature dependence is indicative of enthalpy–entropy compensation determining the equilibrium distributions of the three species. Whereas the pseudoknotted conformation is enthalpically favored (8 bp), it is entropically destabilized with respect to the hairpin structures that have long unstructured regions.Figure 3.

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