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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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Realization of the presented approach as exemplified on two bistable RNAs (4 and 5). (A) Bistable 20 nt RNA 4 with the two competing folds 4′ and 4′′. The red U denotes the 2′-O-13CH3-uridine label. (B) Detection of the folding equilibrium of conformation 4′ and 4′′ by analysis of the imino proton region of the 1H NMR spectrum. (C) 1H, 13C-HSQC of RNA sequence 4. The two folding states give rise to two well-resolved peaks in the HSQC spectrum. Assignment was achieved by means of truncated reference sequences (Supplementary Information). (D) Bistable 32 nt RNA 5 with the two competing folds 5′ and 5′′. The red U denotes the 2′-O-13CH3-uridine label. (E) Severe resonance overlap is found in the imino proton region of the 1H NMR spectrum. (F) 1H, 13C-HSQC spectrum of RNA sequence 5. The two conformations are nicely resolved in the HSQC spectrum. Fold assignment was achieved using a truncated reference sequence (S5a, see Supplementary Information). Conditions: 0.8–1.0 mM RNA, 50 mM sodium phosphate, pH 6.5, H2O/D2O 9/1, 298 K.
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Figure 1: Realization of the presented approach as exemplified on two bistable RNAs (4 and 5). (A) Bistable 20 nt RNA 4 with the two competing folds 4′ and 4′′. The red U denotes the 2′-O-13CH3-uridine label. (B) Detection of the folding equilibrium of conformation 4′ and 4′′ by analysis of the imino proton region of the 1H NMR spectrum. (C) 1H, 13C-HSQC of RNA sequence 4. The two folding states give rise to two well-resolved peaks in the HSQC spectrum. Assignment was achieved by means of truncated reference sequences (Supplementary Information). (D) Bistable 32 nt RNA 5 with the two competing folds 5′ and 5′′. The red U denotes the 2′-O-13CH3-uridine label. (E) Severe resonance overlap is found in the imino proton region of the 1H NMR spectrum. (F) 1H, 13C-HSQC spectrum of RNA sequence 5. The two conformations are nicely resolved in the HSQC spectrum. Fold assignment was achieved using a truncated reference sequence (S5a, see Supplementary Information). Conditions: 0.8–1.0 mM RNA, 50 mM sodium phosphate, pH 6.5, H2O/D2O 9/1, 298 K.

Mentions: The realization of the concept was demonstrated by introducing the 13C-uridine 3 into the 20 nt bistable RNA sequence 4 at position 18 (Figure 1A) (12). We observed two distinct signal patterns in both the imino proton regions of the 1H-NMR spectrum and in the 1H, 13C-HSQC spectrum of sequence 4 originating from the two competing folds, 4′ and 4′′, indicating that the two species refold slowly on the NMR chemical shift time-scale (Figure 1B and C). This nicely demonstrates the sensitivity of the isotope label to its magnetic environment. The 13C, 1H resonances were assigned to their respective conformations by the aid of truncated reference sequences (sequences S4a and S4b, Supplementary Figure S1). Quantification of the equilibrium fold distribution was checked on one hand by the comparative imino proton method [for a detailed description of the comparative imino proton method see reference (8)] and on the other hand by integration of the 1H, 13C-HSQC peaks. Both methods reported the two states to be equally populated, in line with earlier findings (8,12). Importantly, the 13C-modification also proved to be minimally invasive as no population differences compared to the wild-type sequence were found. This was further supported by UV-melting experiments, which show that the 2′-O-methoxy modification change the melting transition by only 1–2 K (Supplementary Figure S3) (8).Figure 1.


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)

Realization of the presented approach as exemplified on two bistable RNAs (4 and 5). (A) Bistable 20 nt RNA 4 with the two competing folds 4′ and 4′′. The red U denotes the 2′-O-13CH3-uridine label. (B) Detection of the folding equilibrium of conformation 4′ and 4′′ by analysis of the imino proton region of the 1H NMR spectrum. (C) 1H, 13C-HSQC of RNA sequence 4. The two folding states give rise to two well-resolved peaks in the HSQC spectrum. Assignment was achieved by means of truncated reference sequences (Supplementary Information). (D) Bistable 32 nt RNA 5 with the two competing folds 5′ and 5′′. The red U denotes the 2′-O-13CH3-uridine label. (E) Severe resonance overlap is found in the imino proton region of the 1H NMR spectrum. (F) 1H, 13C-HSQC spectrum of RNA sequence 5. The two conformations are nicely resolved in the HSQC spectrum. Fold assignment was achieved using a truncated reference sequence (S5a, see Supplementary Information). Conditions: 0.8–1.0 mM RNA, 50 mM sodium phosphate, pH 6.5, H2O/D2O 9/1, 298 K.
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Figure 1: Realization of the presented approach as exemplified on two bistable RNAs (4 and 5). (A) Bistable 20 nt RNA 4 with the two competing folds 4′ and 4′′. The red U denotes the 2′-O-13CH3-uridine label. (B) Detection of the folding equilibrium of conformation 4′ and 4′′ by analysis of the imino proton region of the 1H NMR spectrum. (C) 1H, 13C-HSQC of RNA sequence 4. The two folding states give rise to two well-resolved peaks in the HSQC spectrum. Assignment was achieved by means of truncated reference sequences (Supplementary Information). (D) Bistable 32 nt RNA 5 with the two competing folds 5′ and 5′′. The red U denotes the 2′-O-13CH3-uridine label. (E) Severe resonance overlap is found in the imino proton region of the 1H NMR spectrum. (F) 1H, 13C-HSQC spectrum of RNA sequence 5. The two conformations are nicely resolved in the HSQC spectrum. Fold assignment was achieved using a truncated reference sequence (S5a, see Supplementary Information). Conditions: 0.8–1.0 mM RNA, 50 mM sodium phosphate, pH 6.5, H2O/D2O 9/1, 298 K.
Mentions: The realization of the concept was demonstrated by introducing the 13C-uridine 3 into the 20 nt bistable RNA sequence 4 at position 18 (Figure 1A) (12). We observed two distinct signal patterns in both the imino proton regions of the 1H-NMR spectrum and in the 1H, 13C-HSQC spectrum of sequence 4 originating from the two competing folds, 4′ and 4′′, indicating that the two species refold slowly on the NMR chemical shift time-scale (Figure 1B and C). This nicely demonstrates the sensitivity of the isotope label to its magnetic environment. The 13C, 1H resonances were assigned to their respective conformations by the aid of truncated reference sequences (sequences S4a and S4b, Supplementary Figure S1). Quantification of the equilibrium fold distribution was checked on one hand by the comparative imino proton method [for a detailed description of the comparative imino proton method see reference (8)] and on the other hand by integration of the 1H, 13C-HSQC peaks. Both methods reported the two states to be equally populated, in line with earlier findings (8,12). Importantly, the 13C-modification also proved to be minimally invasive as no population differences compared to the wild-type sequence were found. This was further supported by UV-melting experiments, which show that the 2′-O-methoxy modification change the melting transition by only 1–2 K (Supplementary Figure S3) (8).Figure 1.

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