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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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(A) Multi-stable RNA sequence 6 is able to fold into a pseudoknotted conformation 6′ and two hairpin structures 6′′ and 6′′′. Truncated reference sequences 6a and 6b mimic fold 6′′ and 6′′′, respectively. The red U denotes the 2′-O-13CH3-uridine label. (B) Imino proton region of the 1H NMR spectrum of sequence 6 and references 6a and 6b. The detection of multiple conformations of RNA 6 is hampered due to NH resonance overlap originating from the individual folds. Asterisks denote unassigned peaks most likely originating from fold 6′′ and 6′′′. (C) 1H, 13C-HSQC of RNA sequence 6. The three possible folding states can be easily detected and assigned in the HSQC spectrum. Fold assignment was achieved with truncated reference sequences 6a and 6b. Here, an overlay of the HSQC spectra of 6a and 6b is shown. 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 2: (A) Multi-stable RNA sequence 6 is able to fold into a pseudoknotted conformation 6′ and two hairpin structures 6′′ and 6′′′. Truncated reference sequences 6a and 6b mimic fold 6′′ and 6′′′, respectively. The red U denotes the 2′-O-13CH3-uridine label. (B) Imino proton region of the 1H NMR spectrum of sequence 6 and references 6a and 6b. The detection of multiple conformations of RNA 6 is hampered due to NH resonance overlap originating from the individual folds. Asterisks denote unassigned peaks most likely originating from fold 6′′ and 6′′′. (C) 1H, 13C-HSQC of RNA sequence 6. The three possible folding states can be easily detected and assigned in the HSQC spectrum. Fold assignment was achieved with truncated reference sequences 6a and 6b. Here, an overlay of the HSQC spectra of 6a and 6b is shown. Conditions: 0.75 mM RNA, 2.5 mM MgCl2, 50 mM sodium phosphate, pH 6.5, H2O/D2O 9/1, 298 K.

Mentions: As a final example RNA sequence 6 was chosen (18,19). The oligonucleotide 6 coexists in three competing folds, a pseudoknotted conformation 6′, a 5′-hairpin fold 6′′ and a 3′-hairpin fold 6′′′ (Figure 2A). The presence of multiple folding states is not directly observable by the imino proton resonances, as the NH resonances originating from all three conformations (Ψ-knot sequence 6′, 5′-hairpin 6′′ and 3′-hairpin 6′′′) are strongly overlapping (Figure 2B).Figure 2.


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)

(A) Multi-stable RNA sequence 6 is able to fold into a pseudoknotted conformation 6′ and two hairpin structures 6′′ and 6′′′. Truncated reference sequences 6a and 6b mimic fold 6′′ and 6′′′, respectively. The red U denotes the 2′-O-13CH3-uridine label. (B) Imino proton region of the 1H NMR spectrum of sequence 6 and references 6a and 6b. The detection of multiple conformations of RNA 6 is hampered due to NH resonance overlap originating from the individual folds. Asterisks denote unassigned peaks most likely originating from fold 6′′ and 6′′′. (C) 1H, 13C-HSQC of RNA sequence 6. The three possible folding states can be easily detected and assigned in the HSQC spectrum. Fold assignment was achieved with truncated reference sequences 6a and 6b. Here, an overlay of the HSQC spectra of 6a and 6b is shown. 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 2: (A) Multi-stable RNA sequence 6 is able to fold into a pseudoknotted conformation 6′ and two hairpin structures 6′′ and 6′′′. Truncated reference sequences 6a and 6b mimic fold 6′′ and 6′′′, respectively. The red U denotes the 2′-O-13CH3-uridine label. (B) Imino proton region of the 1H NMR spectrum of sequence 6 and references 6a and 6b. The detection of multiple conformations of RNA 6 is hampered due to NH resonance overlap originating from the individual folds. Asterisks denote unassigned peaks most likely originating from fold 6′′ and 6′′′. (C) 1H, 13C-HSQC of RNA sequence 6. The three possible folding states can be easily detected and assigned in the HSQC spectrum. Fold assignment was achieved with truncated reference sequences 6a and 6b. Here, an overlay of the HSQC spectra of 6a and 6b is shown. Conditions: 0.75 mM RNA, 2.5 mM MgCl2, 50 mM sodium phosphate, pH 6.5, H2O/D2O 9/1, 298 K.
Mentions: As a final example RNA sequence 6 was chosen (18,19). The oligonucleotide 6 coexists in three competing folds, a pseudoknotted conformation 6′, a 5′-hairpin fold 6′′ and a 3′-hairpin fold 6′′′ (Figure 2A). The presence of multiple folding states is not directly observable by the imino proton resonances, as the NH resonances originating from all three conformations (Ψ-knot sequence 6′, 5′-hairpin 6′′ and 3′-hairpin 6′′′) are strongly overlapping (Figure 2B).Figure 2.

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