Danny Incarnato

RNA‐targeting small molecules are emerging as promising therapeutic modalities, but their development requires methods that define binding sites and evaluate RNA target selectivity. Existing approaches for detecting ligand–RNA interactions have provided powerful foundations, yet many rely on direct cross‐linking or covalent‐capture chemistries whose performance depends on ligand‐specific probe design, warhead compatibility, and local reaction geometry. Here, we report Singlet Oxygen footprinting on RNA in a Ligand‐Directed manner for Mutational Profiling (SOLiD‐MaP), a photochemical platform achieving the labeling resolution for small molecule binding site identification. Using the Mango‐II aptamer and thiazole orange derivatives as a model system, we establish aniline as an efficient nucleophile for singlet oxygen‐mediated RNA labeling and demonstrate target‐selective labeling driven by ligand‐localized photosensitization. We further show that labeling selectivity can be tuned by chemically constraining the singlet oxygen diffusion with a quencher. Finally, we develop a pairwise reverse transcription stop assay and a mutational profiling with next‐generation sequencing readouts to infer ligand‐proximal regions and unambiguously map binding sites. We further extended binding‐region inference to a cellular context. SOLiD‐MaP provides a new, orthogonal strategy for studying small molecule‐RNA recognition and should support RNA‐focused mechanism‐of‐action studies.

RNA‐targeting small molecules are emerging as promising therapeutic modalities, but their development requires methods that define binding sites and evaluate RNA target selectivity. Existing approaches for detecting ligand–RNA interactions have provided powerful foundations, yet many rely on direct cross‐linking or covalent‐capture chemistries whose performance depends on ligand‐specific probe design, warhead compatibility, and local reaction geometry. Here, we report Singlet Oxygen footprinting on RNA in a Ligand‐Directed manner for Mutational Profiling (SOLiD‐MaP), a photochemical platform achieving the labeling resolution for small molecule binding site identification. Using the Mango‐II aptamer and thiazole orange derivatives as a model system, we establish aniline as an efficient nucleophile for singlet oxygen‐mediated RNA labeling and demonstrate target‐selective labeling driven by ligand‐localized photosensitization. We further show that labeling selectivity can be tuned by chemically constraining the singlet oxygen diffusion with a quencher. Finally, we develop a pairwise reverse transcription stop assay and a mutational profiling with next‐generation sequencing readouts to infer ligand‐proximal regions and unambiguously map binding sites. We further extended binding‐region inference to a cellular context. SOLiD‐MaP provides a new, orthogonal strategy for studying small molecule‐RNA recognition and should support RNA‐focused mechanism‐of‐action studies.

Viral RNA genomes contain structured elements that recruit the machinery necessary for their replication. However, these mechanisms remain poorly understood because of RNA structure’s dynamic nature. Using cryo-EM and single-molecule Förster resonance energy transfer, we show that the flaviviral stem-loop A (SLA)—which recruits the viral polymerase NS5 to initiate negative-strand synthesis—populates a conserved, multi-state conformational ensemble governed by a dynamic three-dimensional (3D) motif. Structures of Zika and dengue SLA–NS5 complexes reveal that NS5 engages a preorganized SLA conformation. Notably, destabilizing this conformation impairs negative-strand synthesis without affecting NS5 binding. Thus, the conserved SLA 3D ensemble guides a post-binding conformational search that converts an initial encounter complex into a productive replication-initiation complex—revealing how RNA conformational ensembles, rather than static structures, dictate function. SUMMARY: Viral genomes encode dynamic RNA structures that are specifically recognized by proteins to regulate critical steps of the viral life cycle. Although these RNAs exist as multi-state conformational ensembles, structural studies have largely captured single conformations, limiting our mechanistic understanding of RNA-protein recognition and function. Here, using single-particle cryo-electron microscopy (cryo-EM), single-molecule Förster resonance energy transfer, and biochemical assays, we show how flaviviral genomes use structural dynamics encoded in their stem-loop A (SLA) to guide a conserved, multi-step mechanism of negative-strand synthesis initiation by non-structural protein 5 (NS5). Cryo-EM ensembles of SLAs from dengue, Zika, West Nile, and yellow fever viruses reveal a conserved conformational landscape dictated by alternative stacking configurations of a central three-way junction (3WJ). Cryo-EM structures of SLA-NS5 complexes from dengue and Zika show that NS5 engages a specific state within SLA 3D ensemble, one in which the 3WJ adopts a GRR/A tetraloop-like motif that we identify across diverse structural RNAs. Strikingly, mutations that substantially destabilize this state impair negative-strand synthesis without affecting NS5 binding—decoupling binding from function and ruling out a simple conformational-capture mechanism. Instead, the SLA’s intrinsic structural dynamics direct a post-binding conformational search that converts an initial, conformationally heterogeneous encounter complex into a productive replication-initiation complex. These results demonstrate a mechanism of RNA-protein recognition in which the 3D conformational ensemble of an RNA guides formation of the functional state downstream of binding, and identify a ubiquitous, intrinsically dynamic 3D motif within the SLA’s 3WJ as a critical determinant of flavivirus negative-strand synthesis.

RNA-targeting small molecules are emerging as promising therapeutic modalities, but their development requires methods that define binding sites and evaluate RNA target selectivity. Existing approaches for detecting ligand-RNA interactions have provided powerful foundations, yet many rely on direct crosslinking or covalent-capture chemistries whose performance depends on ligand-specific probe design, warhead compatibility, and local reaction geometry. Here, we report Singlet Oxygen footprinting on RNA in a Ligand-Directed manner for Mutational Profiling (SOLiD-MaP), a photoproximity labelling platform for small molecule-RNA interaction analysis. Using the Mango-II aptamer and thiazole orange derivatives as a model system, we establish aniline as an efficient nucleophile for singlet oxygen-mediated RNA labelling and demonstrate target-selective labelling driven by ligand-localized photosensitization. We further show that labelling selectivity can be tuned by chemically constraining the singlet oxygen diffusion with a quencher. Finally, we develop a pairwise reverse transcription stop assay and a mutational profiling with next-generation sequencing readouts to infer ligand-proximal regions and unambiguously map binding sites. SOLiD-MaP provides a new, orthogonal strategy for studying small molecule-RNA recognition and should support RNA-focused mechanism-of-action studies.

Targeting RNA with small molecules offers a strategy to modulate gene expression at undruggable targets. Traditional screens favor thermodynamically stable, low-entropy RNA motifs with defined conformations, yet these provide limited energetic leverage for functional modulation. Many RNAs instead sample dynamic structural ensembles that small molecules can repartition. Using group I self-splicing introns as a model, we identified the antineoplastic drug Mitoxantrone as a competitive inhibitor of RNA self-splicing (IC₅₀ = 4.3 μM) that stabilizes the native conformation of the T4 td intron. Structure-activity analysis showed that the anthraquinone scaffold alone is insufficient, and basic amine-containing side chains are required for RNA structural modulation. Transcriptome-wide chemical probing in human cells revealed preferential binding to GC-rich structured regions, although only a subset showed structural change. Furthermore, global analysis of 5′ UTR ensembles showed altered structural heterogeneity and translation, demonstrating functional repartitioning of RNA conformational landscapes.

Butyrate, a short-chain fatty acid (SCFA) produced by microbial fermentation of dietary fiber, exerts beneficial metabolic and immunomodulatory functions through hyperactivation of the histone acetylase (HAT) p300 and inhibition of histone deacetylases (HDAC). These effects are widely believed to result in the acetylation of distinct histones that regulate specific genes, including at transcription starting sites (TSS) and enhancers. However, we show that this is not the case, as butyrate dose-dependently increases the acetylation of histones throughout the entire genome. Molecular dynamics simulations suggest that the RING-loop prevents the recruitment of p300 to acetylated histones through its bromodomain, and thereby cannot maintain the acetylation of specific histones through positive feedback. This was confirmed by showing that only catalytically inactive p300 stably binds to acetylated histones. Thus, the epigenetic regulation of specific genes by butyrate is limited, but butyrate instead increases histone acetylation globally resulting in opening of the entire chromatin structure.

Deciphering how nucleotides in genomes encode regulatory instructions and molecular machines is a long-standing goal. Genomic language models (gLMs) implicitly capture functional elements and their organization from genomic sequences alone by modeling probabilities of each nucleotide given its sequence context. However, discovering functional genomic elements from gLMs has been challenging due to the lack of interpretable methods. Here we introduce nucleotide dependencies, which quantify how nucleotide substitutions at one genomic position affect the probabilities of nucleotides at other positions. We demonstrate that nucleotide dependencies are more effective at indicating the deleteriousness of genetic variants than alignment-based conservation and gLM reconstruction. Dependency analysis accurately detects regulatory motifs and highlights bases in contact within RNAs, including pseudoknots and tertiary structure contacts, revealing new, experimentally validated RNA structures. Finally, we leverage dependency maps to reveal critical limitations of several gLM architectures and training strategies. Altogether, nucleotide dependency analysis opens a new avenue for discovering and studying functional elements and their interactions in genomes.

To facilitate the design of RNA-targeted covalent probes we report a bottom-up, structure-informed approach using mutational profiling. We designed a compact library of covalent probes based on the scaffold of Ribocil, a selective inhibitor targeting the bacterial FMN riboswitch and screened them against the FMN riboswitch from Fusobacterium nucleatum, Bacillus subtilis, Escherichia coli and Staphylococcus aureus. This yielded Covacil, a highly base-selective probe that covalently modifies the FMN riboswitch at low micromolar concentrations, within 10 minutes. We validate the site-selectivity and covalency of the probe by competitive photo-affinity labelling and mass-spectrometry. When compared to non-targeted covalent probes, Covacil displayed >1,000-fold increased selectivity towards a specific base within the FMN riboswitch. Finally, we apply Covacil to total RNA and demonstrate that it maintains its base-selective reactivity for the FMN riboswitch within the entire transcriptome.

RNA molecules can populate ensembles of alternative structural conformations; however, comprehensively mapping RNA conformational landscapes within living cells presents notable challenges and has, as such, so far remained elusive. Here, we generate transcriptome-scale maps of RNA secondary structure ensembles in both Escherichia coli and human cells, uncovering features of structurally heterogeneous regions. By combining ensemble deconvolution and covariation analyses, we report the discovery of several bacterial RNA thermometers in the 5′ untranslated regions (UTRs) of the cspG, cspI, cpxP and lpxP mRNAs of Escherichia coli. We mechanistically characterize how these thermometers switch structure in response to cold shock and reveal the CspE chaperone-mediated regulation of lpxP. Furthermore, we introduce a method for the transcriptome-scale mapping of 5′ UTR structures in eukaryotes and leverage it to uncover RNA structural switches regulating the differential usage of open reading frames in the 5′ UTRs of the CKS2 and TXNL4A mRNAs in HEK293 cells. Collectively, this work reveals the complexity of RNA structural dynamics in living cells and provides a resource to accelerate the discovery of regulatory RNA switches.