Excited States of Nucleic Acids Probed by Proton Relaxation Dispersion NMR Spectroscopy

Abstract

In this work an improved stable isotope labeling protocol for nucleic acids is introduced. The novel building blocks eliminate/minimize homonuclear 13C and 1H scalar couplings thus allowing proton relaxation dispersion (RD) experiments to report accurately on the chemical exchange of nucleic acids. Using site‐specific 2H and 13C labeling, spin topologies are introduced into DNA and RNA that make 1H relaxation dispersion experiments applicable in a straightforward manner. The novel RNA/DNA building blocks were successfully incorporated into two nucleic acids. The A‐site RNA was previously shown to undergo a two site exchange process in the micro‐ to millisecond time regime. Using proton relaxation dispersion experiments the exchange parameters determined earlier could be recapitulated, thus validating the proposed approach. We further investigated the dynamics of the cTAR DNA, a DNA transcript that is involved in the viral replication cycle of HIV‐1. Again, an exchange process could be characterized and quantified. This shows the general applicablility of the novel labeling scheme for 1H RD experiments of nucleic acids.

RNA Dynamics by NMR Spectroscopy

“…In the absence of large homonuclear coupling between H5-H6, it was possible to apply a 1 Hv ersion of CPMG, enabling the characterization of a4%E Sw ith al ifetime of 0.5 ms, where chemical shifta nalysisp ointed towards a base-pair reshuffling process. [84] The 5-13 Cu ridine and 2,8-13 C adeninel abels were used for studies of the 21-mer T. thermophila group Ir ibozyme. The group detected as econdary structure switching event with al ifetime of 1msa nd ap opulation of 9%.…”

“…This procedure has been used for 1 HC PMG data of RNA. [84] In addition different analytical solutions are available for different exchange regimes to fit CPMG data. However,c are has to be taken to choose the right analytical expression for the investigated exchange process.…”

Chemical synthesis and NMR spectroscopy of long stable isotope labelled RNA

“…10,12 Thus, the fully chemical SI-labelling protocol can be regarded as an expedient expansion to the settled enzymatic procedures to freely chose the number and positioning of SI-labeled residues into a target RNA. In our hands, however, the standard solid phase synthesis methods are not that well suited to produce larger amounts (450 nmol) and purities higher than 95% for RNAs exceeding 60 nts.…”

“…30 Further, the isolated spin pair 1 H-X (X = 13 C/ 15 N) topologies of the building blocks 1 to 6 minimize the scalar coupling interactions and relaxation pathways and thus make the application of relaxation based NMR experiments, such as relaxation dispersion or CEST experiments, straightforward to probe functional dynamics in RNAs. 10,12,13 Besides, we anticipate a potential use of the large SI labelled RNAs in recently reported mass spectrometric methods to localize protein binding sites, which give valuable information for the 3D structure modelling of large RNP particles. 31,32 We currently also focus on the synthesis of per-deuterated RNA building blocks.…”

Capturing Excited States in the Fast‐Intermediate Exchange Limit in Biological Systems Using 1H NMR Spectroscopy

“…Importantly aw ider range of exchange rates can be covered (with SL up to 20 kHz (Bruker), exchange rates up to 50 kHz can be covered), thus allowing us to characterize conformational exchange faster than 10 kHz accurately in RNAfor the first time. [7] Thep ulse sequence ( Figure 2) is built on ideas from Lundstrçm, [8] Weininger, [6d] Eichmüller, [5] and Hansen. 1 Hc hemical shifts are very sensitive to structural changes,a nd many studies have been dedicated to correlating them to secondary and tertiary structures.…”