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A kinetic model is proposed to predict the probability of dG•dT misincorporation across different polymerases, and provides mechanisms for sequence-dependent misincorporation.

dG•dT and rG•rU ‘wobble’ mispairs in DNA and RNA transiently form base pairs with Watson–Crick geometry via tautomerization and ionization with probabilities that correlate with misincorporation probabilities during replication and translation.

This study develops an NMR-based approach that can capture previously inaccessible, highly transient, low-populated ‘excited states’ in RNA; the localized rearrangements in base-pairing giving rise to these states are found to affect function by changing the exposure of residues required for a specific biological process.

The standard view of the genome is that the two DNA strands are linked by Watson–Crick base pairing. Some deviations from this canonical pairing have been observed when DNA is bound to a ligand. This paper now shows that naked DNA itself can transiently adopt a Hoogsteen base-pairing arrangement. This excited state base pairing provides a means to expand the chemistry and structure of DNA, and has implications for the binding of proteins to and repair of DNA.

m⁶A RNA post-transcriptional modification changes RNA hybridization kinetics. Here the authors show that the methylamino group can adopt syn-conformation pairing with uridine with a mismatch-like conformation in RNA duplex. They also develop a quantitative model that predicts how m6A affects the kinetics of hybridization.

The inability of A-form RNA to form Hoogsteen base pairs provides a mechanism for how post-transcriptional modifications can disrupt RNA structure and might help explain why DNA is the molecular choice for storing genetic information.

Determining dynamic ensembles of biomolecules is still challenging. Here the authors present an approach for rapid RNA ensemble determination that combines RNA structure prediction tools and NMR residual dipolar coupling data and use it to determine atomistic ensemble models for a variety of RNAs.

N⁶-Methyladenosine (m⁶A) is a post-transcriptional RNA modification that modulates RNA structure through a destabilization of m⁶A base pairing. Here the authors use NMR and UV melting experiments and show that m⁶A can also stabilize m⁶A–U base pairs and global RNA structure when positioned adjacent to a 5ʹ bulge.

Short-lived RNA folding intermediates have important roles in the folding of RNA. Here, the authors combine ¹⁵N relaxation dispersion NMR with chemical probing to visualise one of these intermediates, and are able to show it is a secondary structural switch, that might help with folding.

DNA is found in a dynamic equilibrium between standard Watson-Crick (WC) base pairs and non-standard Hoogsteen (HG) base pairs. Here the authors describe the influence of echinomycin and actinomycin D ligands binding on the HG-WC base pair dynamics in DNA.

A high-throughput assay that introduces mismatched base pairs into the DNA sequence shows that mismatches can increase transcription factor binding affinity by prepaying some of the energetic cost of distorting the DNA.

Excited conformation state of biomolecule is transient and high-energy conformation state. Here the authors use NMR spectroscopy and computational modeling to reveal the 3D structure of HIV-1 TAR RNA excited conformational state.

An approach for quantifying the similarity between dynamic ensembles of biomolecular structures is described and applied to RNA ensembles studied by nuclear magnetic resonance spectroscopy and molecular dynamics simulations.

Hoogsteen (HG) base pairs occur transiently within DNA and exhibit altered non Watson–Crick (WC)-pairing geometries with the potential to govern sequence-dependent DNA processes. Here, Alvey et al.show that HG base pairing occurs within diverse sequence contexts and define the energetic landscapes that favour WC-to-HG transitions.

Mutating RNA one nucleotide at a time and measuring the impact of this on its chemical reactivity provides a strategy for determining its three-dimensional structure, and from there, hopefully, its function.

This paper reports an NMR approach that enables the three-dimensional movement of two linked RNA helices to be followed for a period up to milliseconds. The two helices move in a highly correlated manner, demonstrating both twisting and bending. Results with different liganded conformations demonstrate that the motions of the unstructured RNA maps out positions within the dynamic envelope described by the bound structures.

A bird's-eye view of the higher-order structure of HIV-1's entire RNA genome reveals new motifs in surprising places. Structural biologists can now zoom in on these regions to explore their functions further.

Replicative errors contribute to genetic diversity needed for evolution but in high frequency lead to genomic stability. Here, NMR is used to show via a kinetic model that DNA dynamics can determine the misincorporation of A•G and A•8OG mismatches.

Systematic alteration of HIV-1 TAR RNA and quantitative determination of its propensity to bind to the Tat protein establish a key role role for a rare and short-lived RNA state in Tat-dependent transactivation in cells.

The functions of many regulatory RNAs depend on how their 3D structure changes in response to cellular conditions. Recent studies have revealed that RNA exists as a dynamic ensemble of conformations, which form with different probabilities in different cellular conditions and thus modulate RNA function.

AlphaFold 3 represents a breakthrough in predicting the 3D structures of complexes directly from their sequences, offering insights into biomolecular interactions. Extending predictions to molecular behavior and function requires a shift from viewing biomolecules as static 3D structures to dynamic conformational ensembles.

A new way to virtually screen HIV-1 TAR RNA using dynamic ensembles better identifies small molecules targeting RNA secondary structures for development.

Protein-focused lead-identification strategies may be limited in their ability to identify small molecules that bind to cellular RNAs. Docking small molecules against the structural ensemble substantially improves the docking accuracy of TAR and has led to the identification of six new TAR binders, one of which inhibits HIV-1 replication.

In this Review, the authors thoroughly discuss solution-state NMR spectroscopy methods used for characterizing the dynamic structure landscape of RNA molecules, from picosecond to second timescale motions.