Direct 13C-detected NMR experiments for mapping and characterization of hydrogen bonds in RNA

Abstract

In RNA secondary structure determination, it is essential to determine whether a nucleotide is base-paired and not. Base-pairing of nucleotides is mediated by hydrogen bonds. The NMR characterization of hydrogen bonds relies on experiments correlating the NMR resonances of exchangeable protons and can be best performed for structured parts of the RNA, where labile hydrogen atoms are protected from solvent exchange. Functionally important regions in RNA, however, frequently reveal increased dynamic disorder which often leads to NMR signals of exchangeable protons that are broadened beyond (1)H detection. Here, we develop (13)C direct detected experiments to observe all nucleotides in RNA irrespective of whether they are involved in hydrogen bonds or not. Exploiting the self-decoupling of scalar couplings due to the exchange process, the hydrogen bonding behavior of the hydrogen bond donor of each individual nucleotide can be determined. Furthermore, the adaption of HNN-COSY experiments for (13)C direct detection allows correlations of donor-acceptor pairs and the localization of hydrogen-bond acceptor nucleotides. The proposed (13)C direct detected experiments therefore provide information about molecular sites not amenable by conventional proton-detected methods. Such information makes the RNA secondary structure determination by NMR more accurate and helps to validate secondary structure predictions based on bioinformatics.

Coupling between conformational dynamics and catalytic function at the active site of the lead-dependent ribozyme

“…This is particularly true in the case of RNA, for which rugged conformational landscapes are often limiting for function (Williamson 2000;Leulliot and Varani 2001;Perez-Canadillas and Varani 2001;Rambo and Doudna 2004;Al-Hashimi and Walter 2008;Walter 2009). Heteronuclear NMR has emerged as a powerful probe of the conformational dynamics of RNA on a variety of time scales (Bothe et al 2011;Salmon et al 2014), with a particular strength in the detection and characterization of low-population conformers, sampled on the microsecond to millisecond time scale, that may be functionally critical for RNA catalysis and recognition (Hoogstraten et al 2000;Dethoff et al 2012;Ravera et al 2014;Zhao et al 2014;Zhao and Zhang 2015;Furtig et al 2016;Schnieders et al 2017). Given a spectroscopic or biophysical analysis of a molecule's dynamics, true mechanistic insight can in principle be obtained by "dynamics-function" studies, in which a chosen dynamic mode is selectively quenched and the effect on catalysis, binding, or other functional output is assessed.…”

More than Proton Detection—New Avenues for NMR Spectroscopy of RNA

“…The walk through the nucleobase is indicated with a gray dashed line. The figure has been adapted from literature …”

“…B Structural context of the respective uridines (U11 WC base pair, H‐bonded imino proton; U6 sheared GU base pair, sterically shielded imino proton; U7 unpaired nucleotide, fully solvent exposed imino proton) are indicated . The figure has been adapted from the literature …”

“…The figure has been adapted from literature. [42] Figure 6. A Magnetization transfer pathways for 1 H-excited and 13 C-detected 2D experiments that correlate H6 to C6 (black) or H5 to C5 to C4 (red) to measure 1 J(C,C) coupling constants.…”

NMR Spectroscopy of Large Functional RNAs: From Sample Preparation to Low‐Gamma Detection

“…Moreover, we provide protocols for carrying out heteronuclear-detected NMR experiments in general (Support Protocols 7 and 8) and describe how to set-up 13 C-detected NMR experiments for the ribose assignment, namely (H)CC-total correlation spectroscopy (TOCSY), (H)CPC and (H)CPC-HCC-TOCSY experiments (Basic Protocol 3; Richter et al, 2010). Furthermore, a guide to set up a so-called CN-spin filter heteronuclear single quantum coherence (HSQC) experiment for the determination of the status of hydrogen bonding is provided (Basic Protocol 4; Fürtig et al, 2016). For the characterization of amino groups in RNA, we provide a protocol for the 13 C-detected C(N)H-heteronuclear doublequantum correlation (HDQC) experiment (Basic Protocol 5, Support Protocol 9) as well as the "amino"-nuclear Overhauser effect spectroscopy (NOESY) experiment (Basic Protocol 6;Schnieders et al, 2019).…”