Identification of RNA Base Pairs and Complete Assignment of Nucleobase Resonances by Proton‐Detected Solid‐State NMR Spectroscopy at 100 kHz MAS

Abstract

Knowledge of RNA structure, either in isolation or in complex, is fundamental to understand the mechanism of cellular processes. Solid‐state NMR (ssNMR) is applicable to high molecular‐weight complexes and does not require crystallization; thus, it is well‐suited to study RNA as part of large multicomponent assemblies. Recently, we solved the first structures of both RNA and an RNA‐protein complex by ssNMR using conventional 13C‐ and 15N‐detection. This approach is limited by the severe overlap of the RNA peaks together with the low sensitivity of multidimensional experiments. Here, we overcome the limitations in sensitivity and resolution by using 1H‐detection at fast MAS rates. We develop experiments that allow the identification of complete nucleobase spin‐systems together with their site‐specific base pair pattern using sub‐milligram quantities of one uniformly labelled RNA sample. These experiments provide rapid access to RNA secondary structure by ssNMR in protein‐RNA complexes of any size.

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

“…This method provides typical 13 C linewidths of 1 ppm (29mer HIV TAR RNA) (Huang et al, 2012) or even 0.5 ppm for the 26mer box C/D RNA in complex with L7Ae protein (Marchanka et al, 2013;Marchanka et al, 2015). The same complex shows an 1 H linewidth of separated C1′-H1′ resonances in the protonated ribose of 150 Hz (0.15 ppm) by 1 H-detection at 100 kHz MAS on a 1 GHz spectrometer, while the 1 H linewidth of imino resonances on an 850 MHz spectrometer estimates to 200-300 Hz (0.23-0.35 ppm) (Aguion et al, 2021). In the 1 H-detected imino spectrum of 26mer DIS-HIV-1 RNA acquired at 40 kHz MAS, the linewidths of the 1 H and 15 N dimensions are equal to 500 Hz (0.9 ppm) and 70 Hz (1 ppm), respectively (Yang et al, 2017).…”

“…Very recently, we have addressed this challenge and have published the set of ssNMR experiments that allow assignment of nucleobase resonances (Aguion et al, 2021).…”

“…As in 13 C-detected ssNMR, assignment of these resonances remains challenging, but N3 resonances of cytidines can likewise be obtained indirectly through base-paired guanosines. Here, the close distance of cytidine N3 and H1 resonances in the basepaired guanosine (r 2.0 Å) can be exploited in a simple 2D 1 H-15 N-HSQC experiment with long-range 1 H-15 N/ 15 N-1 H CP transfer times of 8 ms (Aguion et al, 2021).…”

“…Our and Carlomagno's laboratories have also been active in the characterization of protein-RNA interfaces, and we have recently determined the structure of the same protein-RNA complex by a combination of paramagnetic relaxation enhancement (PRE)-aided ssNMR and chemical shift perturbation (CSP) analysis (Ahmed et al, 2020). 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).…”

“…Unfortunately, the low resolution in the 1 H dimension (∼500 Hz for imino protons) did not allow the identification of different spin systems and thus the unambiguous assignment of hydrogen bonds. In our recent study at 100 kHz MAS, in view of good linewidths in the proton dimension (200-300 Hz), spin system-specific detection of base pairs was possible (Aguion et al, 2021), allowing rapid identification of almost all base pairs present in 26mer box C/D RNA. While the 2D/3D (H)N(HH)NH experiment detects WC G:C and non-WC U:U base pairs, the 2D/3D (H)N(HH)CH experiment identifies WC A:U and non-WC G:A base pairs.…”