Hashim M. Al-Hashimi

RNA dynamics play a fundamental role in many cellular functions. However, there is no general framework to describe these complex processes, which typically consist of many structural maneuvers that occur over timescales ranging from picoseconds to seconds. Here, we classify RNA dynamics into distinct modes representing transitions between basins on a hierarchical free-energy landscape. These transitions include large-scale secondary-structural transitions at >0.1-s timescales, base-pair/tertiary dynamics at microsecond-to-millisecond timescales, stacking dynamics at timescales ranging from nanoseconds to microseconds, and other “jittering” motions at timescales ranging from picoseconds to nanoseconds. We review various modes within these three different tiers, the different mechanisms by which they are used to regulate function, and how they can be coupled together to achieve greater functional complexity.

Conformational changes in nucleic acids play a key role in the way genetic information is stored, transferred, and processed in living cells. Here, we describe new approaches that employ a broad range of experimental data, including NMR-derived chemical shifts and residual dipolar couplings, small-angle X-ray scattering, and computational approaches such as molecular dynamics simulations to determine ensembles of DNA and RNA at atomic resolution. We review the complementary information that can be obtained from diverse sets of data and the various methods that have been developed to combine these data with computational methods to construct ensembles and assess their uncertainty. We conclude by surveying RNA and DNA ensembles determined using these methods, highlighting the unique physical and functional insights obtained so far.

Dimethyl sulfoxide (DMSO) is widely used as a cosolvent to solubilize hydrophobic compounds in RNA-ligand binding assays. Although it is known that high concentrations of DMSO (>75%) can significantly affect RNA structure and folding energetics, a thorough analysis of how lower concentrations (<10%) of DMSO typically used in binding assays affects RNA structure and ligand binding has not been undertaken. Here, we use NMR and 2-aminopurine fluorescence spectroscopy to examine how DMSO affects the structure, dynamics, and ligand binding properties of two flexible hairpin RNAs: the transactivation response element from HIV-1 and bacterial ribosomal A-site. In both cases, 5–10% DMSO decreased stacking interactions and increased local disorder in noncanonical residues within bulges and loops and resulted in 0.3–4-fold reduction in the measured binding affinities for different small molecules, with the greatest reduction observed for an intercalating compound that binds RNA nonspecifically. Our results suggest that, by competing for hydrophobic interactions, DMSO can have a small but significant effect on RNA structure and ligand binding. These effects should be considered when developing ligand binding assays and high throughput screens.

In 1957, a unique pattern of hydrogen bonding between N3 and O4 on uracil and N7 and N6 on adenine was proposed to explain how poly(rU) strands can associate with poly(rA)‐poly(rU) duplexes to form triplexes. Two years later, Karst Hoogsteen visualized such a noncanonical A–T base‐pair through X‐ray analysis of co‐crystals containing 9‐methyladenine and 1‐methylthymine. Subsequent X‐ray analyses of guanine and cytosine derivatives yielded the expected Watson–Crick base‐pairing, but those of adenine and thymine (or uridine) did not yield Watson–Crick base‐pairs, instead favoring “Hoogsteen” base‐pairing. More than two decades ensued without experimental “proof” for A–T Watson–Crick base‐pairs, while Hoogsteen base‐pairs continued to surface in AT‐rich sequences, closing base‐pairs of apical loops, in structures of DNA bound to antibiotics and proteins, damaged and chemically modified DNA, and in polymerases that replicate DNA via Hoogsteen pairing. Recently, NMR studies have shown that base‐pairs in duplex DNA exist as a dynamic equilibrium between Watson–Crick and Hoogsteen forms. There is now little doubt that Hoogsteen base‐pairs exist in significant abundance in genomic DNA, where they can expand the structural and functional versatility of duplex DNA beyond that which can be achieved based only on Watson–Crick base‐pairing. Here, we provide a historical account of the discovery and characterization of Hoogsteen base‐pairs, hoping that this will inform future studies exploring the occurrence and functional importance of these alternative base‐pairs. © 2013 Wiley Periodicals, Inc. Biopolymers 99: 955–968, 2013.

The ability to modulate alignment and measure multiple independent sets of NMR residual dipolar couplings (RDCs) has made it possible to characterize internal motions in proteins at atomic resolution and with time scale sensitivity ranging from picoseconds up to milliseconds. The application of such methods to the study of RNA dynamics, however, remains fundamentally limited by the inability to modulate alignment and by strong couplings between internal and overall motions that complicate the quantitative interpretation of RDCs. Here, we address this problem by showing that RNA alignment can be generally modulated, in a controlled manner, by variable elongation of A-form helices and that the information contained within the measured RDCs can be extracted even in the presence of strong couplings between motions and overall alignment via structure-based prediction of alignment. Using this approach, four RDC data sets, and a broad conformational pool obtained from a 8.2 μs molecular dynamics simulation, we successfully construct and validate an atomic resolution ensemble of human immunodeficiency virus type I transactivation response element RNA. This ensemble reveals local motions in and around the bulge involving changes in stacking and hydrogen-bonding interactions, which are undetectable by traditional spin relaxation and drive global changes in interhelical orientation. This new approach broadens the scope of using RDCs in characterizing the dynamics of nucleic acids.

G·C Hoogsteen base pairs can form transiently in duplex DNA and play important roles in DNA recognition, replication, and repair. G·C Hoogsteen base pairs are thought to be stabilized by protonation of cytosine N3, which affords a second key hydrogen bond, but experimental evidence for this is sparse because the proton cannot be directly visualized by X-ray crystallography and nuclear magnetic resonance spectroscopy. Here, we combine NMR and constant pH molecular dynamics simulations to directly investigate the pK_a of cytosine N3 in a chemically trapped N1-methyl-G·C Hoogsteen base pair within duplex DNA. Analysis of NMR chemical shift perturbations and NOESY data as a function of pH revealed that cytosine deprotonation is coupled to a syn-to-anti transition in N1-methyl-G, which results in a distorted Watson–Crick geometry at pH >9. A four-state analysis of the pH titration profiles yields a lower bound pK_a estimate of 7.2 ± 0.1 for the G·C Hoogsteen base pair, which is in good agreement with the pK_a value (7.1 ± 0.1) calculated independently using constant pH MD simulations. Based on these results and pH-dependent NMR relaxation dispersion measurements, we estimate that under physiological pH (pH 7–8), G·C Hoogsteen base pairs in naked DNA have a population of 0.02–0.002%, as compared to 0.4% for A·T Hoogsteen base pairs, and likely exist primarily as protonated species.

The development of methods for predicting NMR chemical shifts with high accuracy and speed is increasingly allowing use of these abundant, readily accessible measurements in determining the structure and dynamics of proteins. For nucleic acids, however, despite the availability of semiempirical methods for predicting ¹H chemical shifts, their use in determining the structure and dynamics has not yet been examined. Here, we show that ¹H chemical shifts offer powerful restraints for RNA structure determination, allowing discrimination of native structure from non-native states to within 2–4 Å, and 1H chemical shifts can provide valuable information for constructing RNA dynamic ensembles, large uncertainties in the chemical shift predictions and inherent degeneracies lead to higher uncertainties as compared to residual dipolar couplings.

The visualization of RNA conformational changes has provided fundamental insights into how regulatory RNAs carry out their biological functions. The RNA structural transitions that have been characterized so far involve long-lived species that can be captured by structure characterization techniques. Here we report the nuclear magnetic resonance visualization of RNA transitions towards ‘invisible’ excited states (ESs), which exist in too little abundance (2–13%) and for too short a duration (45–250 μs) to allow structural characterization by conventional techniques. Transitions towards ESs result in localized rearrangements in base-pairing that alter building block elements of RNA architecture, including helix–junction–helix motifs and apical loops. The ES can inhibit function by sequestering residues involved in recognition and signalling or promote ATP-independent strand exchange. Thus, RNAs do not adopt a single conformation, but rather exist in rapid equilibrium with alternative ESs, which can be stabilized by cellular cues to affect functional outcomes.

Sequence-specific DNA flexibility plays a key role in a variety of cellular interactions that are critical for gene packaging, expression, and regulation, yet few studies have experimentally explored the sequence dependence of DNA dynamics that occur on biologically relevant time scales. Here, we use nuclear magnetic resonance (NMR) carbon spin relaxation combined with molecular dynamics (MD) simulations to examine the picosecond to nanosecond dynamics in a variety of dinucleotide steps as well as in varying length homopolymeric A_n·T_n repeats (A_n-tracts, where n = 2, 4, or 6) that exhibit unusual structural and mechanical properties. We extend the NMR spin relaxation time scale sensitivity deeper into the nanosecond regime by using glycerol and a longer DNA duplex to slow overall tumbling. Our studies reveal a structurally unique A-tract core (for n > 3) that is uniformly rigid, flanked by junction steps that show increasing sugar flexibility with A-tract length. High sugar mobility is observed at pyrimidine residues at the A-tract junctions, which is encoded at the dinucleotide level (CA, TG, and CG steps) and increases with A-tract length. The MD simulations reproduce many of these trends, particularly the overall rigidity of A-tract base and sugar sites, and suggest that the sugar–backbone dynamics could involve transitions in sugar pucker and phosphate backbone BI ↔ BII equilibria. Our results reinforce an emerging view that sequence-specific DNA flexibility can be imprinted in dynamics occurring deep within the nanosecond time regime that is difficult to characterize experimentally at the atomic level. Such large-amplitude sequence-dependent backbone fluctuations might flag the genome for specific DNA recognition.