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 13C exchange NMR spectroscopy. For this purpose a straightforward labeling technique was elaborated using a 2′-13C-methoxy uridine modification, which was prepared by a two-step synthesis and introduced into RNA using standard protocols. Using 13C longitudinal exchange NMR spectroscopy the refolding kinetics of a 20 nt bistable RNA were characterized at temperatures between 298 and 310 K, 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 13C relaxation dispersion NMR spectroscopy.

Kinetics of DNA Refolding from Longitudinal Exchange NMR Spectroscopy

“…Longitudinal exchange experiments are based on pulse sequences previously published for 15 N, yielding 13 C, 1 H 2D correlation maps with amplitude modulation of correlation-and exchange-peaks determined by longitudinal exchange rate constants and the kinetics of interconversion. [8,15] For a sample (0.8 mm) of sequence 6, arrays of spectra were recorded at 306, 309, 312, and 315 K with mixing periods of 10, 50, 100, 200, 300, 400, 600, and 800 ms (10,30,60,100,150,250,350,500,600 and 800 ms at 315 K). The size of the data matrices for each spectrum was 1024 48 complex data points, the number of scans was 128 and the interscan delay was 1.5 s, yielding a total measuring time of 30 h at each temperature.…”

“…[7] NMR spectroscopy has proven to be well suited for detecting and quantifying distinct folding states of nucleic acids, and for determining the kinetics and the thermodynamics of the refolding process. [8] In this work we present a site-specific 13 Cisotope labeling procedure for DNA and use it to characterize secondary structure heterogeneity of a bistable DNA sequence. [7] We introduce isolated 13 C-spin labels into the methyl groups of thymidines that can be employed to detect and quantify dynamic properties using relaxation-based NMR techniques for 13 CH 3 -spin systems.…”

“…[8] Within this work we compare the existing data of bistable RNAs with a related, rationally designed DNA sequence 6, which exists in two different stable hairpin folds with nearly equal population distribution (Figure 1 A). This model system was introduced recently, and its fold heterogeneity and the respective populations were determined by comparative imino proton NMR and fluorine NMR spectroscopy.…”

“…These data favor a transition state model with base pairs of both folds being formed and disrupted at the same time ("associative mechanism"). [8] Refolding kinetics of various bistable RNA systems comprising 20 to 34 nucleotides have been reported. [6b, c, h-j, 8] In Table S2 in the Supporting Information we compare data from these systems to our results of the bistable model DNA sequence 6.…”

NMR Spectroscopic Studies of the Conformational Ensembles of Intrinsically Disordered Proteins

“…(Kloiber et al 2011;Korzhnev and Kay 2008;Korzhnev et al 2004b;Schanda et al 2008) These methods were developed primarily for folded substates. Yet they are applicable for intrinsically disordered proteins, too.…”