Characterizing Hydrogen Bonds in Intact RNA from MS2 Bacteriophage Using Solid State Magic Angle Spinning NMR

Abstract

Ribonucleic acid (RNA) is a polymer with pivotal functions in many biological processes. RNA structure determination is thus a vital step towards understanding its function. The secondary structure of RNA is stabilized by hydrogen bonds formed between nucleotide base pairs and it defines the positions and shapes of functional stem-loops, internal loops, bulges, and other functional and structural elements. In this work we present a methodology for studying large intact RNA molecules using homonuclear 15N solid state nuclear magnetic resonance (NMR) spectroscopy. We show that Proton Driven Spin Diffusion (PDSD) experiments with long mixing times, up to 16s, improved by the incorporation of 1H Radiofrequency Dipolar Recoupling (RFDR) pulses, reveal key hydrogen-bond contacts. In the full-length RNA isolated from MS2 phage, we observed strong and dominant contributions of G-C Watson-Crick base pairs, and beyond these common interactions, we observe a significant contribution of the G-U wobble base pairs. Using the improved technique facilitates characterization of hydrogen-bond types in intact large-scale RNA using solid-state NMR. It can be highly useful to guide secondary structure prediction techniques, and possibly to refine higher resolution structure determination methods.

Strategies for RNA Resonance Assignment by 13C/15N- and 1H-Detected Solid-State NMR Spectroscopy

“…Finally, the first studies on RNA at 40 kHz MAS (Yang et al, 2017;Zhao et al, 2019) and MAS ≥ 100 kHz (Marchanka et al, 2018b;Aguion et al, 2021) have been performed, reporting both assignment of resonances and identification of base pairs by sensitive 1 H-detected ssNMR spectroscopy. In the research field of phage viruses, the Goldbourt laboratory has obtained nucleotide-type assignment for very large native DNA (Sergeyev et al, 2011;Morag et al, 2014;Goldbourt, 2019) and has recently identified base pairs in native 1.2 MDa RNA from the bacteriophage MS2 (Lusky et al, 2021).…”

“…Structural determination of RNA by ssNMR comprises several steps that include sample preparation, spin system-specific (assignment of resonances within a nucleotide spin system) and site-specific/sequential (assignment of defined spin systems to a specific residue within the RNA) assignment of resonances, and the collection of distance and angular restraints which are then used in structural calculations (Marchanka and Carlomagno, 2019) (Figure 1). While in some studies, unambiguous assignment of RNA resonances is not necessary to obtain valuable structural information (Olsen et al, 2005;Huang et al, 2010Huang et al, , 2011Yang et al, 2017;Lusky et al, 2021), in most cases, site-specific assignment of resonances is the main limiting and crucial step in structure determination by NMR.…”

“…Site-specific assignment via imino-imino sequential walk was not possible in 15 N-detected spectra due to poor 15 N chemical shift dispersion and typical 15 N linewidths ≥ 1 ppm. Recently, the Goldbourt laboratory has employed 15 N, 15 N PDSD and 15 N, 15 N PDSD•RFDR experiments to identify the presence of WC G:C and wobble G:U base pairs in native 1 MDa-sized bacteriophage MS2 RNA and obtained nucleotidetype-specific assignments (Lusky et al, 2021). In all 15 N-detected experiments described above, after the initial 1 H-15 N cross-polarization (CP), magnetization is evolved on 15 N during t 1 .…”