Probing RNA dynamics via longitudinal exchange and CPMG relaxation dispersion NMR spectroscopy using a sensitive 13C-methyl label

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.

Scheme 1.

Synthesis of the 2′-O-¹³CH₃-uridine phosphoramidite 3; (a) 6 eq. ¹³C-magnesium methoxide [Mg(O¹³CH₃)₂, freshly prepared from magnesium turnings and ¹³CH₃OH] in anhydrous dimethylformamide, 100°C, 2 h, 93%; (b) 1.5 eq. (2-cyanoethyl)-N,N-diisopropylchlorophosphoramidite, 10 eq. N-ethyldimethylamine in anhydrous dichloromethane, room temperature, 2.5 h, 71%.

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.

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.

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.

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).

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 (k_{4′→4′′}) (solid line) and ln (k_{4′′→4′}) (dashed line) as a function of the inverse temperature. (B) ln (K) [computed as ln (k_{4′→4′′}/k_{4′′→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 k_{4′→4′′}, k_{4′′→4′}, and K.

Figure 6.

¹³C longitudinal exchange experiments conducted on sequence 5. (A) Interconversion between conformations 5′ and 5′′ with the uridine label highlighted in red. (B) Left panel: intensities of the corrrelation peak corresponding to conformation 5′ and exchange peak corresponding to transition 5′ → 5′′ as a function of mixing time. Right panel: correlation peak pertinent to fold 5′′ and exchange peak for the transition 5′′ → 5′. Results at 314 (316) K are depicted as circles (diamonds) and fits at 314 (316) K are shown as solid/dashed lines. Error bars were obtained on the basis of spectral noise in a Monte Carlo analysis.

Figure 7.

Analysis of CPMG relaxation dispersion of RNA sequence 6. A two-state process that is fast on the chemical shift time-scale was fit to the data of conformer 6′ (A) and 6′′′ (B) (for details of the fitting procedure see text and Supplementary Information). Experimental data points are shown as black circles and the fit is depicted as a red line. Spectrometer field strengths are indicated and the secondary structures of the two states are shown as inserts. Both conformers 6′ and 6′′′ exchange with their respective excited state at a rate constant of ∼500 s⁻¹.