Isotope Labels Combined with Solution NMR Spectroscopy Make Visible the Invisible Conformations of Small-to-Large RNAs

Abstract

RNA is central to the proper function of cellular processes important for life on earth and implicated in various medical dysfunctions. Yet, RNA structural biology lags significantly behind that of proteins, limiting mechanistic understanding of RNA chemical biology. Fortunately, solution NMR spectroscopy can probe the structural dynamics of RNA in solution at atomic resolution, opening the door to their functional understanding. However, NMR analysis of RNA, with only four unique ribonucleotide building blocks, suffers from spectral crowding and broad linewidths, especially as RNAs grow in size. One effective strategy to overcome these challenges is to introduce NMR-active stable isotopes into RNA. However, traditional uniform labeling methods introduce scalar and dipolar couplings that complicate the implementation and analysis of NMR measurements. This challenge can be circumvented with selective isotope labeling. In this review, we outline the development of labeling technologies and their application to study biologically relevant RNAs and their complexes ranging in size from 5 to 300 kDa by NMR spectroscopy.

Syntheses of Specifically 15N‐Labeled Adenosine and Guanosine

“…In addition, the 13 C chemical shifts can provide valuable information. Recent work using these labeled DNA or RNA fragments include Dayie, Olenginski, and Taiwo (2022) and Lagoya (2002).…”

“…In addition, the 13 C chemical shifts can provide valuable information. Recent work using these labeled DNA or RNA fragments include Dayie, Olenginski, and Taiwo ( 2022 ); Olenginski, Taiwo, Leblanc, and Dayie ( 2021 ); Olengiinsky (2021); Goldberga et al. ( 2019 ); Becette, Olenginski, and Dayie ( 2019 ); Nussbaumer et al.…”

Synthesis and Enzymatic Incorporation of a Dual-App Nucleotide Probe That Reports Antibiotics-Induced Conformational Change in the Bacterial Ribosomal Decoding Site RNA

“…In order to perform its function, RNA adopts complex secondary and tertiary structures, which are influenced by posttranscriptional modifications, changes in environmental conditions, and interaction with proteins, metabolites, and external ligands. − Chemical tools and biophysical techniques based on fluorescence, NMR, and X-ray crystallography are widely employed to study the structure, dynamics, and recognition properties of RNA. − While fluorescence spectroscopy aids in the rapid analysis of RNA in solution and cells, NMR and X-ray crystallography techniques provide information at the atomic level. These methods require fluorophore-labeled, isotope-enriched, and heavy atom-labeled RNA molecules as basic components of nucleic acids are nonemissive and lack intrinsic labels suitable for such analyses. − In this context, environment-sensitive nucleoside analogs inserted into RNA oligonucleotides (ONs) serve as useful tools to analyze their structure and recognition. − More recently, 19 F NMR has emerged as a valuable biophysical handle to study nucleic acid conformations as the 19 F atom is 100% naturally abundant and its chemical shift is highly environment-sensitive. , Chemically synthesized 19 F-labeled ONs have been employed in studying RNA structures and their interaction with proteins and ligands. − …”

Hydrogen Isotope Separation Using Graphene-Based Membranes in Liquid Water

“…A large amount of heavy water is used as the neutron moderator in nuclear fusion reactors. , It is also widely used in laboratory spectroscopy and dynamics research, such as neutron scattering, − isotope tracing, − and solvent for proton nuclear magnetic resonance spectroscopy . Due to its low abundance (∼156 ppm) in nature, methods to gain heavy water with high purity are urgently required for industrial and research applications .…”