Developments in solution-state NMR yield broader and deeper views of the dynamic ensembles of nucleic acids

Abstract

Nucleic acids do not fold into a single conformation, and dynamic ensembles are needed to describe their propensities to cycle between different conformations when performing cellular functions. We review recent advances in solution-state nuclear magnetic resonance (NMR) methods and their integration with computational techniques that are improving the ability to probe the dynamic ensembles of DNA and RNA. These include computational approaches for predicting chemical shifts from structure and generating conformational libraries from sequence, measurements of exact nuclear Overhauser effects, development of new probes to study chemical exchange using relaxation dispersion, faster and more sensitive real-time NMR techniques, and new NMR approaches to tackle large nucleic acid assemblies. We discuss how these advances are leading to new mechanistic insights into gene expression and regulation.

Keywords: Dynamic ensembles; Excited conformational states; Nucleic acids; Solution state NMR.

Figure 1.

Determining dynamic ensembles of nucleic acids using NMR and computational methods. (a) Shown are NMR-derived dynamic ensembles for RNA (UUCG apical loop [13] and HIV-TAR bulge [14]) and DNA (duplexes [18] and a G-quadruplex [19]). The figures were adapted from original publication. (b) Representative fragments of an RNA hairpin structure used to compute chemical shift using the AF-QM/MM approach (top). The figure was adapted from [22]. Also shown is a comparison between measured and AF-QM/MM predicted ¹³C chemical shifts for a conventional NMR structure and an RDC-derived dynamic ensemble of a DNA duplex (bottom). The data shown in the figure was adapted from [23]. (c) The principle of eNOE. A mobile proton H1 samples positions that are close to both protons H2 and H3. The eNOE data is better explained by a 2-state ensemble shown in blue and red relative to a model which assumes a single-state structure. The figure was adapted from [25]. (d) The Rosetta FARFAR structure prediction algorithm uses an RNA secondary structure to rapidly generate a conformation library which can be used in conjunction with NMR data to determine a dynamic ensemble. The figure was adapted from [14].

Figure 2.

A growing number of nuclei are targeted for relaxation dispersion measurements (CEST, CPMG and R_1ρ) in studies of nucleic acid ESs. The list of probes is growing steadily from an initial focus on protonated carbons, imino and amino (G-N2) nitrogens (highlighted using blue circles) to now include protons, non-protonated and amino (C-N4) nitrogens, ribose C5', and nuclei in modified bases including m⁶A, m⁵C and cmo⁵U (highlighted using red circles). Also shown are the types of RD experiments performed for the various probes.

Figure 3.

NMR reveals RNA and DNA ESs that play important roles in biochemical processes. (a-c) RNA ESs form by reshuffling base pairs. (a) The miR-34a:mRNA duplex undergoes conformational exchange between the GS and the ES, which has an elongated 8-mer seed and altered RNA topology, leading to formation of the active RISC complex. The figure was adapted from [43]. (b) pre-miR-21 forms a pH dependent protonated ES with an A⁺(anti)·G(syn) base pair (bp) that is more efficiently recognized and processed by Dicer. The figure was adapted from [45]. (c) Domain 6 of type II intron forms an ES with a bulge adenine that has a predominantly (>99%) C3' endo sugar pucker conformation, suitable for exon ligation during splicing and reverse splicing. The figure was adapted from [47]. (d) Wobble G·T and G·U mismatches form tautomer (blue) and anionic (green) Watson-Crick-like ES conformations that can evade fidelity checkpoints during DNA replication and cause spontaneous mutations. cmo⁵U increases the population of the anionic Watson-Crick ES conformation. The figure was adapted from [50]. (e) Watson-Crick A·T bps form Hoogsteen ES conformations which increase the damage susceptibility of canonical duplex DNA. (f) The methylamino group of m⁶A forms an anti ES needed for base pairing, thereby slowing down duplex hybridization and conformational transitions.

Figure 4.

Real-time NMR and methods to study large nucleic acid assemblies. (a) Schematic representation of the interconverting transcriptional intermediates of 2′dG-sensing riboswitch at transcript lengths 113 and 137 nt with and without ligand. Ligand binding slows the conformational exchange between the OFF and ON state below the transcription rate, triggering the OFF state function. The figure was adapted from [60]. (b) 1D imino spectra of the guanine-sensing riboswitch (GSR) aptamer domain (GSR^apt) of the Bacillus subtilis xpt-pbuX operon with (red) and without (blue) injection of hyperpolarized water (HyperW). The figure was adapted from [62]. (c) The 2D ¹³C-¹H HMQC spectrum of a nucleosome core particle (NCP) (shown is a cartoon representation of NCP, PDB ID 6ESF), in which the 153-bp Widom DNA is highly deuterated with ¹³CH₃-methyl labeled at five sites indicated by black circles. The methyl group resonances belonging to the DNA and protein components are shown in red and gray, respectively, with the positions of the m⁶A methyls aliased (*). The figure was adapted from [65]. (d) Recently developed ¹⁹F labeling schemes for nucleic acids. (e) 2D ¹⁹F-¹³C TROSY spectra of 5-fluorouracil (red) modified human hepatitis B virus encapsidation signal epsilon (hHBV ε) element. The figure was adapted from [72].