A 300-fold enhancement of imino nucleic acid resonances by hyperpolarized water provides a new window for probing RNA refolding by 1D and 2D NMR

Abstract

NMR sensitivity-enhancement methods involving hyperpolarized water could be of importance for solution-state biophysical investigations. Hyperpolarized water (HyperW) can enhance the 1H NMR signals of exchangeable sites by orders of magnitude over their thermal counterparts, while providing insight into chemical exchange and solvent accessibility at a site-resolved level. As HyperW’s enhancements are achieved by exploiting fast solvent exchanges associated with minimal interscan delays, possibilities for the rapid monitoring of chemical reactions and biomolecular (re)folding are opened. HyperW NMR can also accommodate heteronuclear transfers, facilitating the rapid acquisition of 2-dimensional (2D) 15N-1H NMR correlations, and thereby combining an enhanced spectral resolution with speed and sensitivity. This work demonstrates how these qualities can come together for the study of nucleic acids. HyperW injections were used to target the guanine-sensing riboswitch aptamer domain (GSRapt) of the xpt-pbuX operon in Bacillus subtilis. Unlike what had been observed in proteins, where residues benefited of HyperW NMR only if/when sufficiently exposed to water, these enhancements applied to every imino resonance throughout the RNA. The >300-fold enhancements observed in the resulting 1H NMR spectra allowed us to monitor in real time the changes that GSRapt undergoes upon binding hypoxanthine, a high-affinity interaction leading to conformational refolding on a ∼1-s timescale at 36 °C. Structural responses could be identified for several nucleotides by 1-dimensional (1D) imino 1H NMR as well as by 2D HyperW NMR spectra acquired upon simultaneous injection of hyperpolarized water and hypoxanthine. The folding landscape revealed by this HyperW strategy for GSRapt, is briefly discussed.

Characterization of protein–ligand interactions by SABRE

“…Similar information is available if proteins or nucleic acids receive polarization transfer from previously hyperpolarized water. 10,11 Water protons exchange with labile protons on amide or hydroxy groups in macromolecules. Enhanced signals observed for the corresponding positions provide sequence specic information on solvent exposure and molecular dynamics.…”

Real-time NMR spectroscopy in the study of biomolecular kinetics and dynamics

“…A removable injector allows using smaller sample volumes and minimizing the disturbance of the magnetic field homogeneity. While originally not intended for measuring kinetics, also dissolution DNP (see below) uses rapid mixing to inject polarized water into the NMR tube allowing triggering kinetics by adding a folding cofactor simultaneously with the polarized water, as shown recently (Chen et al, 2013;Novakovic et al, 2020b). Biomacromolecules can be unfolded in many different ways (Fürtig et al, 2007a;Roder et al, 2004).…”

“…In a more recent application, Novakovic et al used dissolution-DNP to monitor the real-time refolding of the RNA aptamer domain of guanine-sensing riboswitch (GSW) upon ligand binding (Novakovic et al, 2020b). Different to proteins, the exchange of solvent water with imino sites in RNAs is sufficiently fast not only in unstructured but also in structured regions of the RNA, and thus, all RNA sites can benefit from increase signal-to-noise due to exchange transfer.…”

Time-resolved NMR-spectroscopic studies of conformational dynamics in DNA G-quadruplexes

“…Further, the rather slow interconversion dynamics harbour the speculation that there might exist proteins with a refolding chaperoning activity in the cell. Newly developed NMR methods (such as ultrafast 2D-NMR 634 in combination with signal enhancement 639,641,768 ) will help to expand the experimental toolkit to tackle this challenge.…”

“…This holds particularly true for multidimensional, mostly heteronuclearcorrelated ( 13 C, 15 N) experiments. Recent advances 629 in acquisition (SOFAST and BEST) [630][631][632][633] and ultrafast methods 634,635 , sensitivity enhancement (hyperpolarized water) [636][637][638][639][640] and combinations of ultrafast acquisition with hyperpolarization, 641,642 have narrowed the timeframe of observable real-time dynamics down to seconds. 643,644 Figure 27: The NMR-timescale 602,605,613 , overview of: the relevant molecular motions for nucleic acids in solution, timescale and frequencies with indicated exchange-regime (fast, intermediate, slow), NMR methods and experiments to probe directly specific dynamic processes, and relevant parameters in NMR experiments that influence and modulate the shape and intensity of NMR signals.…”