Characterizing RNA Excited States Using NMR Relaxation Dispersion

Abstract

Changes in RNA secondary structure play fundamental roles in the cellular functions of a growing number of non-coding RNAs. This chapter describes NMR-based approaches for characterizing microsecond-to-millisecond changes in RNA secondary structure that are directed toward short-lived and low-populated species often referred to as “excited states”. Compared to larger-scale changes in RNA secondary structure, transitions towards excited states do not require assistance from chaperones, are often orders of magnitude faster, and are localized to a small number of nearby base pairs in and around non-canonical motifs. Here we describe a procedure for characterizing RNA excited states using off-resonance R1ρ NMR relaxation dispersion utilizing low-to-high spin-lock fields (25–3000 Hz). R1ρ NMR relaxation dispersion experiments are used to measure carbon and nitrogen chemical shifts in base and sugar moieties of the excited state. The chemical shift data is then interpreted with the aid of secondary structure prediction to infer potential excited states that feature alternative secondary structures. Candidate structures are then tested by using mutations, single-atom substitutions, or by changing physiochemical conditions, such as pH and temperature, to either stabilize or destabilize the candidate excited state. The resulting chemical shifts of the mutants or under different physiochemical conditions are then compared to those of the ground and excited state. Application is illustrated with a focus on the transactivation response element (TAR) from the human immune deficiency virus type 1 (HIV-1), which exists in dynamic equilibrium with at least two distinct excited states.

Shortening the HIV-1 TAR RNA Bulge by a Single Nucleotide Preserves Motional Modes over a Broad Range of Time Scales

“…To clearly show exchange parameters, relaxation dispersion profiles are plotted as R 2 + R ex to remove the contributions of R 1 to R 1 ρ , which do not provide any information about chemical exchange. 84 …”

Atomic structures of excited state A–T Hoogsteen base pairs in duplex DNA by combining NMR relaxation dispersion, mutagenesis, and chemical shift calculations

“…This has proven to be an attractive approach for nucleic acids because their ESs often feature non-canonical mismatches that can be stabilized by point substitution mutations or single atom substitutions 10,16,35 . The mutants also provide an opportunity to explore the function of the ESs in vitro and in vivo 36,37 .…”

“…Whether similar changes in sugar-backbone conformation take place when forming ES Hoogsteen bps in unmodified DNA duplexes remains to be established. RD studies 35 have so far relied on base (C2, C5, C6, C8, N1/3) and sugar (C1′) probes that provide limited information regarding sugar-backbone conformation. The C1′ chemical shift is insensitive to phosphodiester torsion angles, and while it is sensitive to sugar pucker, this can be obscured by an even greater sensitivity to the glycosidic Χ angle, which can vary significantly when forming non-canonical bps 47 .…”

A dynamic look backward and forward

“…Work in my own laboratory has focused on spin-relaxation studies of proteins in solution; however, the methods and experimental approaches pioneered in solution NMR spectroscopy have been adapted to nucleic acids in solution [67–69] and to solid-state NMR studies of biological macromolecules [70–72]. These advances suggest that the renaissance in NMR spin relaxation of biological macromolecules that began in 1988 is still vibrant and is likely to generate additional Gunther Laukien Prize awards in the future.…”