DNA mismatches reveal conformational penalties in protein-DNA recognition.

Transcription factors recognize specific genomic sequences to regulate complex gene-expression programs. Although it is well-established that transcription factors bind to specific DNA sequences using a combination of base readout and shape recognition, some fundamental aspects of protein-DNA binding remain poorly understood1,2. Many DNA-binding proteins induce changes in the structure of the DNA outside the intrinsic B-DNA envelope. However, how the energetic cost that is associated with distorting the DNA contributes to recognition has proven difficult to study, because the distorted DNA exists in low abundance in the unbound ensemble3-9. Here we use a high-throughput assay that we term SaMBA (saturation mismatch-binding assay) to investigate the role of DNA conformational penalties in transcription factor-DNA recognition. In SaMBA, mismatched base pairs are introduced to pre-induce structural distortions in the DNA that are much larger than those induced by changes in the Watson-Crick sequence. Notably, approximately 10% of mismatches increased transcription factor binding, and for each of the 22 transcription factors that were examined, at least one mismatch was found that increased the binding affinity. Mismatches also converted non-specific sites into high-affinity sites, and high-affinity sites into 'super sites' that exhibit stronger affinity than any known canonical binding site. Determination of high-resolution X-ray structures, combined with nuclear magnetic resonance measurements and structural analyses, showed that many of the DNA mismatches that increase binding induce distortions that are similar to those induced by protein binding-thus prepaying some of the energetic cost incurred from deforming the DNA. Our work indicates that conformational penalties are a major determinant of protein-DNA recognition, and reveals mechanisms by which mismatches can recruit transcription factors and thus modulate replication and repair activities in the cell10,11.

CasPEDIA Database: a functional classification system for class 2 CRISPR-Cas enzymes.

CRISPR-Cas enzymes enable RNA-guided bacterial immunity and are widely used for biotechnological applications including genome editing. In particular, the Class 2 CRISPR-associated enzymes (Cas9, Cas12 and Cas13 families), have been deployed for numerous research, clinical and agricultural applications. However, the immense genetic and biochemical diversity of these proteins in the public domain poses a barrier for researchers seeking to leverage their activities. We present CasPEDIA (http://caspedia.org), the Cas Protein Effector Database of Information and Assessment, a curated encyclopedia that integrates enzymatic classification for hundreds of different Cas enzymes across 27 phylogenetic groups spanning the Cas9, Cas12 and Cas13 families, as well as evolutionarily related IscB and TnpB proteins. All enzymes in CasPEDIA were annotated with a standard workflow based on their primary nuclease activity, target requirements and guide-RNA design constraints. Our functional classification scheme, CasID, is described alongside current phylogenetic classification, allowing users to search related orthologs by enzymatic function and sequence similarity. CasPEDIA is a comprehensive data portal that summarizes and contextualizes enzymatic properties of widely used Cas enzymes, equipping users with valuable resources to foster biotechnological development. CasPEDIA complements phylogenetic Cas nomenclature and enables researchers to leverage the multi-faceted nucleic-acid targeting rules of diverse Class 2 Cas enzymes.

Dynamic basis for dA•dGTP and dA•d8OGTP misincorporation via Hoogsteen base pairs.

Replicative errors contribute to the genetic diversity needed for evolution but in high frequency can lead to genomic instability. Here, we show that DNA dynamics determine the frequency of misincorporating the A•G mismatch, and altered dynamics explain the high frequency of 8-oxoguanine (8OG) A•8OG misincorporation. NMR measurements revealed that Aanti•Ganti (population (pop.) of >91%) transiently forms sparsely populated and short-lived Aanti+•Gsyn (pop. of ~2% and kex = kforward + kreverse of ~137 s-1) and Asyn•Ganti (pop. of ~6% and kex of ~2,200 s-1) Hoogsteen conformations. 8OG redistributed the ensemble, rendering Aanti•8OGsyn the dominant state. A kinetic model in which Aanti+•Gsyn is misincorporated quantitatively predicted the dA•dGTP misincorporation kinetics by human polymerase ß, the pH dependence of misincorporation and the impact of the 8OG lesion. Thus, 8OG increases replicative errors relative to G because oxidation of guanine redistributes the ensemble in favor of the mutagenic Aanti•8OGsyn Hoogsteen state, which exists transiently and in low abundance in the A•G mismatch.

Eukaryotic RNA-guided endonucleases evolved from a unique clade of bacterial enzymes.

RNA-guided endonucleases form the crux of diverse biological processes and technologies, including adaptive immunity, transposition, and genome editing. Some of these enzymes are components of insertion sequences (IS) in the IS200/IS605 and IS607 transposon families. Both IS families encode a TnpA transposase and a TnpB nuclease, an RNA-guided enzyme ancestral to CRISPR-Cas12s. In eukaryotes, TnpB homologs occur as two distinct types, Fanzor1s and Fanzor2s. We analyzed the evolutionary relationships between prokaryotic TnpBs and eukaryotic Fanzors, which revealed that both Fanzor1s and Fanzor2s stem from a single lineage of IS607 TnpBs with unusual active site arrangement. The widespread nature of Fanzors implies that the properties of this particular lineage of IS607 TnpBs were particularly suited to adaptation in eukaryotes. Biochemical analysis of an IS607 TnpB and Fanzor1s revealed common strategies employed by TnpBs and Fanzors to co-evolve with their cognate transposases. Collectively, our results provide a new model of sequential evolution from IS607 TnpBs to Fanzor2s, and Fanzor2s to Fanzor1s that details how genes of prokaryotic origin evolve to give rise to new protein families in eukaryotes.

Measuring thermodynamic preferences to form non-native conformations in nucleic acids using ultraviolet melting.

Thermodynamic preferences to form non-native conformations are crucial for understanding how nucleic acids fold and function. However, they are difficult to measure experimentally because this requires accurately determining the population of minor low-abundance (<10%) conformations in a sea of other conformations. Here, we show that melting experiments enable facile measurements of thermodynamic preferences to adopt nonnative conformations in DNA and RNA. The key to this "delta-melt" approach is to use chemical modifications to render specific minor non-native conformations the major state. The validity and robustness of delta-melt is established for four different non-native conformations under various physiological conditions and sequence contexts through independent measurements of thermodynamic preferences using NMR. Delta-melt is faster relative to NMR, simple, and cost-effective and enables thermodynamic preferences to be measured for exceptionally low-populated conformations. Using delta-melt, we obtained rare insights into conformational cooperativity, obtaining evidence for significant cooperativity (1.0 to 2.5 kcal/mol) when simultaneously forming two adjacent Hoogsteen base pairs. We also measured the thermodynamic preferences to form G-C+ and A-T Hoogsteen and A-T base open states for nearly all 16 trinucleotide sequence contexts and found distinct sequence-specific variations on the order of 2 to 3 kcal/mol. This rich landscape of sequence-specific non-native minor conformations in the DNA double helix may help shape the sequence specificity of DNA biochemistry. Thus, melting experiments can now be used to access thermodynamic information regarding regions of the free energy landscape of biomolecules beyond the native folded and unfolded conformations.

Probing Watson-Crick and Hoogsteen base pairing in duplex DNA using dynamic nuclear polarization solid-state NMR spectroscopy.

The majority of base pairs in double-stranded DNA exist in the canonical Watson-Crick geometry. However, they can also adopt alternate Hoogsteen conformations in various complexes of DNA with proteins and small molecules, which are key for biological function and mechanism. While detection of Hoogsteen base pairs in large DNA complexes and assemblies poses considerable challenges for traditional structural biology techniques, we show here that multidimensional dynamic nuclear polarization-enhanced solid-state NMR can serve as a unique spectroscopic tool for observing and distinguishing Watson-Crick and Hoogsteen base pairs in a broad range of DNA systems based on characteristic NMR chemical shifts and internuclear dipolar couplings. We illustrate this approach using a model 12-mer DNA duplex, free and in complex with the antibiotic echinomycin, which features two central adenine-thymine base pairs with Watson-Crick and Hoogsteen geometry, respectively, and subsequently extend it to the ∼200 kDa Widom 601 DNA nucleosome core particle.

Improved genome editing by an engineered CRISPR-Cas12a.

CRISPR-Cas12a is an RNA-guided, programmable genome editing enzyme found within bacterial adaptive immune pathways. Unlike CRISPR-Cas9, Cas12a uses only a single catalytic site to both cleave target double-stranded DNA (dsDNA) (cis-activity) and indiscriminately degrade single-stranded DNA (ssDNA) (trans-activity). To investigate how the relative potency of cis- versus trans-DNase activity affects Cas12a-mediated genome editing, we first used structure-guided engineering to generate variants of Lachnospiraceae bacterium Cas12a that selectively disrupt trans-activity. The resulting engineered mutant with the biggest differential between cis- and trans-DNase activity in vitro showed minimal genome editing activity in human cells, motivating a second set of experiments using directed evolution to generate additional mutants with robust genome editing activity. Notably, these engineered and evolved mutants had enhanced ability to induce homology-directed repair (HDR) editing by 2-18-fold compared to wild-type Cas12a when using HDR donors containing mismatches with crRNA at the PAM-distal region. Finally, a site-specific reversion mutation produced improved Cas12a (iCas12a) variants with superior genome editing efficiency at genomic sites that are difficult to edit using wild-type Cas12a. This strategy establishes a pipeline for creating improved genome editing tools by combining structural insights with randomization and selection. The available structures of other CRISPR-Cas enzymes will enable this strategy to be applied to improve the efficacy of other genome-editing proteins.

Kinetic Resolution of the Atomic 3D Structures Formed by Ground and Excited Conformational States in an RNA Dynamic Ensemble.

Knowing the 3D structures formed by the various conformations populating the RNA free-energy landscape, their relative abundance, and kinetic interconversion rates is required to obtain a quantitative and predictive understanding of how RNAs fold and function at the atomic level. While methods integrating ensemble-averaged experimental data with computational modeling are helping define the most abundant conformations in RNA ensembles, elucidating their kinetic rates of interconversion and determining the 3D structures of sparsely populated short-lived RNA excited conformational states (ESs) remains challenging. Here, we developed an approach integrating Rosetta-FARFAR RNA structure prediction with NMR residual dipolar couplings and relaxation dispersion that simultaneously determines the 3D structures formed by the ground-state (GS) and ES subensembles, their relative abundance, and kinetic rates of interconversion. The approach is demonstrated on HIV-1 TAR, whose six-nucleotide apical loop was previously shown to form a sparsely populated (∼13%) short-lived (lifetime ∼ 45 µs) ES. In the GS, the apical loop forms a broad distribution of open conformations interconverting on the pico-to-nanosecond time scale. Most residues are unpaired and preorganized to bind the Tat-superelongation protein complex. The apical loop zips up in the ES, forming a narrow distribution of closed conformations, which sequester critical residues required for protein recognition. Our work introduces an approach for determining the 3D ensemble models formed by sparsely populated RNA conformational states, provides a rare atomic view of an RNA ES, and kinetically resolves the atomic 3D structures of RNA conformational substates, interchanging on time scales spanning 6 orders of magnitude, from picoseconds to microseconds.

Understanding the characteristics of nonspecific binding of drug-like compounds to canonical stem-loop RNAs and their implications for functional cellular assays.

Identifying small molecules that selectively bind an RNA target while discriminating against all other cellular RNAs is an important challenge in RNA-targeted drug discovery. Much effort has been directed toward identifying drug-like small molecules that minimize electrostatic and stacking interactions that lead to nonspecific binding of aminoglycosides and intercalators to many stem-loop RNAs. Many such compounds have been reported to bind RNAs and inhibit their cellular activities. However, target engagement and cellular selectivity assays are not routinely performed, and it is often unclear whether functional activity directly results from specific binding to the target RNA. Here, we examined the propensities of three drug-like compounds, previously shown to bind and inhibit the cellular activities of distinct stem-loop RNAs, to bind and inhibit the cellular activities of two unrelated HIV-1 stem-loop RNAs: the transactivation response element (TAR) and the rev response element stem IIB (RREIIB). All compounds bound TAR and RREIIB in vitro, and two inhibited TAR-dependent transactivation and RRE-dependent viral export in cell-based assays while also exhibiting off-target interactions consistent with nonspecific activity. A survey of X-ray and NMR structures of RNA-small molecule complexes revealed that aminoglycosides and drug-like molecules form hydrogen bonds with functional groups commonly accessible in canonical stem-loop RNA motifs, in contrast to ligands that specifically bind riboswitches. Our results demonstrate that drug-like molecules can nonspecifically bind stem-loop RNAs most likely through hydrogen bonding and electrostatic interactions and reinforce the importance of assaying for off-target interactions and RNA selectivity in vitro and in cells when assessing novel RNA-binders.

Revealing A-T and G-C Hoogsteen base pairs in stressed protein-bound duplex DNA.

Watson-Crick base pairs (bps) are the fundamental unit of genetic information and the building blocks of the DNA double helix. However, A-T and G-C can also form alternative 'Hoogsteen' bps, expanding the functional complexity of DNA. We developed 'Hoog-finder', which uses structural fingerprints to rapidly screen Hoogsteen bps, which may have been mismodeled as Watson-Crick in crystal structures of protein-DNA complexes. We uncovered 17 Hoogsteen bps, 7 of which were in complex with 6 proteins never before shown to bind Hoogsteen bps. The Hoogsteen bps occur near mismatches, nicks and lesions and some appear to participate in recognition and damage repair. Our results suggest a potentially broad role for Hoogsteen bps in stressed regions of the genome and call for a community-wide effort to identify these bps in current and future crystal structures of DNA and its complexes.

2'-O-Methylation can increase the abundance and lifetime of alternative RNA conformational states.

2'-O-Methyl (Nm) is a highly abundant post-transcriptional RNA modification that plays important biological roles through mechanisms that are not entirely understood. There is evidence that Nm can alter the biological activities of RNAs by biasing the ribose sugar pucker equilibrium toward the C3'-endo conformation formed in canonical duplexes. However, little is known about how Nm might more broadly alter the dynamic ensembles of flexible RNAs containing bulges and internal loops. Here, using NMR and the HIV-1 transactivation response (TAR) element as a model system, we show that Nm preferentially stabilizes alternative secondary structures in which the Nm-modified nucleotides are paired, increasing both the abundance and lifetime of low-populated short-lived excited states by up to 10-fold. The extent of stabilization increased with number of Nm modifications and was also dependent on Mg2+. Through phi-value analysis, the Nm modification also provided rare insights into the structure of the transition state for conformational exchange. Our results suggest that Nm could alter the biological activities of Nm-modified RNAs by modulating their secondary structural ensembles as well as establish the utility of Nm as a tool for the discovery and characterization of RNA excited state conformations.

Hoogsteen base pairs increase the susceptibility of double-stranded DNA to cytotoxic damage.

As the Watson-Crick faces of nucleobases are protected in dsDNA, it is commonly assumed that deleterious alkylation damage to the Watson-Crick faces of nucleobases predominantly occurs when DNA becomes single-stranded during replication and transcription. However, damage to the Watson-Crick faces of nucleobases has been reported in dsDNA in vitro through mechanisms that are not understood. In addition, the extent of protection from methylation damage conferred by dsDNA relative to ssDNA has not been quantified. Watson-Crick base pairs in dsDNA exist in dynamic equilibrium with Hoogsteen base pairs that expose the Watson-Crick faces of purine nucleobases to solvent. Whether this can influence the damage susceptibility of dsDNA remains unknown. Using dot-blot and primer extension assays, we measured the susceptibility of adenine-N1 to methylation by dimethyl sulfate (DMS) when in an A-T Watson-Crick versus Hoogsteen conformation. Relative to unpaired adenines in a bulge, Watson-Crick A-T base pairs in dsDNA only conferred ∼130-fold protection against adenine-N1 methylation, and this protection was reduced to ∼40-fold for A(syn)-T Hoogsteen base pairs embedded in a DNA-drug complex. Our results indicate that Watson-Crick faces of nucleobases are accessible to alkylating agents in canonical dsDNA and that Hoogsteen base pairs increase this accessibility. Given the higher abundance of dsDNA relative to ssDNA, these results suggest that dsDNA could be a substantial source of cytotoxic damage. The work establishes DMS probing as a method for characterizing A(syn)-T Hoogsteen base pairs in vitro and also lays the foundation for a sequencing approach to map A(syn)-T Hoogsteen and unpaired adenines genome-wide in vivo.

Probing conformational transitions towards mutagenic Watson-Crick-like G·T mismatches using off-resonance sugar carbon R1ρ relaxation dispersion.

NMR off-resonance R1ρ relaxation dispersion measurements on base carbon and nitrogen nuclei have revealed that wobble G·T/U mismatches in DNA and RNA duplexes exist in dynamic equilibrium with short-lived, low-abundance, and mutagenic Watson-Crick-like conformations. As Watson-Crick-like G·T mismatches have base pairing geometries similar to Watson-Crick base pairs, we hypothesized that they would mimic Watson-Crick base pairs with respect to the sugar-backbone conformation as well. Using off-resonance R1ρ measurements targeting the sugar C3' and C4' nuclei, a structure survey, and molecular dynamics simulations, we show that wobble G·T mismatches adopt sugar-backbone conformations that deviate from the canonical Watson-Crick conformation and that transitions toward tautomeric and anionic Watson-Crick-like G·T mismatches restore the canonical Watson-Crick sugar-backbone. These measurements also reveal kinetic isotope effects for tautomerization in D2O versus H2O, which provide experimental evidence in support of a transition state involving proton transfer. The results provide additional evidence in support of mutagenic Watson-Crick-like G·T mismatches, help rule out alternative inverted wobble conformations in the case of anionic G·T-, and also establish sugar carbons as new non-exchangeable probes of this exchange process.

Increasing the length of poly-pyrimidine bulges broadens RNA conformational ensembles with minimal impact on stacking energetics.

Helical elements separated by bulges frequently undergo transitions between unstacked and coaxially stacked conformations during the folding and function of noncoding RNAs. Here, we examine the dynamic properties of poly-pyrimidine bulges of varying length (n = 1-4, 7) across a range of Mg2+ concentrations using HIV-1 TAR RNA as a model system and solution NMR spectroscopy. In the absence of Mg2+, helices linked by bulges with n ≥ 3 residues adopt predominantly unstacked conformations (stacked population <15%), whereas one-bulge and two-bulge motifs adopt predominantly stacked conformations (stacked population >74%). In the presence of 3 mM Mg2+, the helices predominantly coaxially stack (stacked population >84%), regardless of bulge length, and the midpoint for the Mg2+-dependent stacking transition is within threefold regardless of bulge length. In the absence of Mg2+, the difference between free energy of interhelical coaxial stacking across the bulge variants is estimated to be ∼2.9 kcal/mol, based on an NMR chemical shift mapping with stacking being more energetically disfavored for the longer bulges. This difference decreases to ∼0.4 kcal/mol in the presence of Mg2+ NMR RDCs and resonance intensity data show increased dynamics in the stacked state with increasing bulge length in the presence of Mg2+ We propose that Mg2+ helps to neutralize the growing electrostatic repulsion in the stacked state with increasing bulge length thereby increasing the number of coaxial conformations that are sampled. Energetically compensated interhelical stacking dynamics may help to maximize the conformational adaptability of RNA and allow a wide range of conformations to be optimally stabilized by proteins and ligands.

Why are Hoogsteen base pairs energetically disfavored in A-RNA compared to B-DNA?

A(syn)-U/T and G(syn)-C+ Hoogsteen (HG) base pairs (bps) are energetically more disfavored relative to Watson-Crick (WC) bps in A-RNA as compared to B-DNA by >1 kcal/mol for reasons that are not fully understood. Here, we used NMR spectroscopy, optical melting experiments, molecular dynamics simulations and modified nucleotides to identify factors that contribute to this destabilization of HG bps in A-RNA. Removing the 2'-hydroxyl at single purine nucleotides in A-RNA duplexes did not stabilize HG bps relative to WC. In contrast, loosening the A-form geometry using a bulge in A-RNA reduced the energy cost of forming HG bps at the flanking sites to B-DNA levels. A structural and thermodynamic analysis of purine-purine HG mismatches reveals that compared to B-DNA, the A-form geometry disfavors syn purines by 1.5-4 kcal/mol due to sugar-backbone rearrangements needed to sterically accommodate the syn base. Based on MD simulations, an additional penalty of 3-4 kcal/mol applies for purine-pyrimidine HG bps due to the higher energetic cost associated with moving the bases to form hydrogen bonds in A-RNA versus B-DNA. These results provide insights into a fundamental difference between A-RNA and B-DNA duplexes with important implications for how they respond to damage and post-transcriptional modifications.

Extending the Sensitivity of CEST NMR Spectroscopy to Micro-to-Millisecond Dynamics in Nucleic Acids Using High-Power Radio-Frequency Fields.

Biomolecules undergo motions on the micro-to-millisecond timescale to adopt low-populated transient states that play important roles in folding, recognition, and catalysis. NMR techniques, such as Carr-Purcell-Meiboom-Gill (CPMG), chemical exchange saturation transfer (CEST), and R1ρ are the most commonly used methods for characterizing such transitions at atomic resolution under solution conditions. CPMG and CEST are most effective at characterizing motions on the millisecond timescale. While some implementations of the R1ρ experiment are more broadly sensitive to motions on the micro-to-millisecond timescale, they entail the use of selective irradiation schemes and inefficient 1D data acquisition methods. Herein, we show that high-power radio-frequency fields can be used in CEST experiments to extend the sensitivity to faster motions on the micro-to-millisecond timescale. Given the ease of implementing high-power fields in CEST, this should make it easier to characterize micro-to-millisecond dynamics in biomolecules.

NMR Chemical Exchange Measurements Reveal That N6-Methyladenosine Slows RNA Annealing.

N6-Methyladenosine (m6A) is an abundant epitranscriptomic modification that plays important roles in many aspects of RNA metabolism. While m6A is thought to mainly function by recruiting reader proteins to specific RNA sites, the modification can also reshape RNA-protein and RNA-RNA interactions by altering RNA structure mainly by destabilizing base pairing. Little is known about how m6A and other epitranscriptomic modifications might affect the kinetic rates of RNA folding and other conformational transitions that are also important for cellular activity. Here, we used NMR R1ρ relaxation dispersion and chemical exchange saturation transfer to noninvasively and site-specifically measure nucleic acid hybridization kinetics. The methodology was validated on two DNA duplexes and then applied to examine how a single m6A alters the hybridization kinetics in two RNA duplexes. The results show that m6A minimally impacts the rate constant for duplex dissociation, changing koff by ∼1-fold but significantly slows the rate of duplex annealing, decreasing kon by ∼7-fold. A reduction in the annealing rate was observed robustly for two different sequence contexts at different temperatures, both in the presence and absence of Mg2+. We propose that rotation of the N6-methyl group from the preferred syn conformation in the unpaired nucleotide to the energetically disfavored anti conformation required for Watson-Crick pairing is responsible for the reduced annealing rate. The results help explain why in mRNA m6A slows down tRNA selection and more generally suggest that m6A may exert cellular functions by reshaping the kinetics of RNA conformational transitions.

Insights into Watson-Crick/Hoogsteen breathing dynamics and damage repair from the solution structure and dynamic ensemble of DNA duplexes containing m1A.

In the canonical DNA double helix, Watson-Crick (WC) base pairs (bps) exist in dynamic equilibrium with sparsely populated (∼0.02-0.4%) and short-lived (lifetimes ∼0.2-2.5 ms) Hoogsteen (HG) bps. To gain insights into transient HG bps, we used solution-state nuclear magnetic resonance spectroscopy, including measurements of residual dipolar couplings and molecular dynamics simulations, to examine how a single HG bp trapped using the N1-methylated adenine (m1A) lesion affects the structural and dynamic properties of two duplexes. The solution structure and dynamic ensembles of the duplexes reveals that in both cases, m1A forms a m1A•T HG bp, which is accompanied by local and global structural and dynamic perturbations in the double helix. These include a bias toward the BI backbone conformation; sugar repuckering, major-groove directed kinking (∼9°); and local melting of neighboring WC bps. These results provide atomic insights into WC/HG breathing dynamics in unmodified DNA duplexes as well as identify structural and dynamic signatures that could play roles in m1A recognition and repair.

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

NMR relaxation dispersion studies indicate that in canonical duplex DNA, Watson-Crick base pairs (bps) exist in dynamic equilibrium with short-lived low abundance excited state Hoogsteen bps. N1-methylated adenine (m1A) and guanine (m1G) are naturally occurring forms of damage that stabilize Hoogsteen bps in duplex DNA. NMR dynamic ensembles of DNA duplexes with m1A-T Hoogsteen bps reveal significant changes in sugar pucker and backbone angles in and around the Hoogsteen bp, as well as kinking of the duplex towards the major groove. Whether these structural changes also occur upon forming excited state Hoogsteen bps in unmodified duplexes remains to be established because prior relaxation dispersion probes provided limited information regarding the sugar-backbone conformation. Here, we demonstrate measurements of C3' and C4' spin relaxation in the rotating frame (R1ρ) in uniformly 13C/15N labeled DNA as sensitive probes of the sugar-backbone conformation in DNA excited states. The chemical shifts, combined with structure-based predictions using an automated fragmentation quantum mechanics/molecular mechanics method, show that the dynamic ensemble of DNA duplexes containing m1A-T Hoogsteen bps accurately model the excited state Hoogsteen conformation in two different sequence contexts. Formation of excited state A-T Hoogsteen bps is accompanied by changes in sugar-backbone conformation that allow the flipped syn adenine to form hydrogen-bonds with its partner thymine and this in turn results in overall kinking of the DNA toward the major groove. Results support the assignment of Hoogsteen bps as the excited state observed in canonical duplex DNA, provide an atomic view of DNA dynamics linked to formation of Hoogsteen bps, and lay the groundwork for a potentially general strategy for solving structures of nucleic acid excited states.

RNA language models predict mutations that improve RNA function.

Structured RNA lies at the heart of many central biological processes, from gene expression to catalysis. While advances in deep learning enable the prediction of accurate protein structural models, RNA structure prediction is not possible at present due to a lack of abundant high-quality reference data. Furthermore, available sequence data are generally not associated with organismal phenotypes that could inform RNA function. We created GARNET (Gtdb Acquired RNa with Environmental Temperatures), a new database for RNA structural and functional analysis anchored to the Genome Taxonomy Database (GTDB). GARNET links RNA sequences derived from GTDB genomes to experimental and predicted optimal growth temperatures of GTDB reference organisms. This enables construction of deep and diverse RNA sequence alignments to be used for machine learning. Using GARNET, we define the minimal requirements for a sequence- and structure-aware RNA generative model. We also develop a GPT-like language model for RNA in which triplet tokenization provides optimal encoding. Leveraging hyperthermophilic RNAs in GARNET and these RNA generative models, we identified mutations in ribosomal RNA that confer increased thermostability to the Escherichia coli ribosome. The GTDB-derived data and deep learning models presented here provide a foundation for understanding the connections between RNA sequence, structure, and function.