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Riboswitches are highly structured RNA regulators of gene expression. Although found in all three domains of life, they are particularly abundant and widespread in bacteria, including many human pathogens, thus making them an attractive target for antimicrobial development. Moreover, the functional versatility of riboswitches to recognize a myriad of ligands, including ions, amino acids, and diverse small-molecule metabolites, has enabled the generation of synthetic aptamers that have been used as molecular probes, sensors, and regulatory RNA devices. Generally speaking, a riboswitch consists of a ligand-sensing aptamer domain and an expression platform, whose genetic control is achieved through the formation of mutually exclusive secondary structures in a ligand-dependent manner. For most riboswitches, this involves formation of the aptamer’s P1 helix and the regulation of its stability, whose competing structure turns gene expression ON/OFF at the level of transcription or translation. Structural knowledge of the conformational changes involving the P1 regulatory helix, therefore, is essential in understanding the structural basis for ligand-induced conformational switching. This review provides a summary of riboswitch cases for which ligand-free and ligand-bound structures have been determined. Comparative analyses of these structures illustrate the uniqueness of these riboswitches, not only in ligand sensing but also in the various structural mechanisms used to achieve the same end of regulating switch helix stability. In all cases, the ligand stabilizes the P1 helix primarily through coaxial stacking interactions that promote helical continuity.

Amino acid availability in Gram-positive bacteria is monitored by T-box riboswitches. T-boxes directly bind tRNAs, assess their aminoacylation state, and regulate the transcription or translation of downstream genes to maintain nutritional homeostasis. Here, we report cocrystal and cryo-EM structures of Geobacillus kaustophilus and Bacillus subtilis T-box–tRNA complexes, detailing their multivalent, exquisitely selective interactions. The T-box forms a U-shaped molecular vise that clamps the tRNA, captures its 3′ end using an elaborate ‘discriminator’ structure, and interrogates its aminoacylation state using a steric filter fashioned from a wobble base pair. In the absence of aminoacylation, T-boxes clutch tRNAs and form a continuously stacked central spine, permitting transcriptional readthrough or translation initiation. A modeled aminoacyl disrupts tRNA-T-box stacking, severing the central spine and blocking gene expression. Our data establish a universal mechanism of amino acid sensing on tRNAs and gene regulation by T-box riboswitches and exemplify how higher-order RNA-RNA interactions achieve multivalency and specificity.

RNA flexibility is reflected in its heterogeneous conformation. Through direct visualization using atomic force microscopy (AFM) and the adenosylcobalamin riboswitch aptamer domain as an example, we show that a single RNA sequence folds into conformationally and architecturally heterogeneous structures under near-physiological solution conditions. Recapitulated 3D topological structures from AFM molecular surfaces reveal that all conformers share the same secondary structural elements. Only a population-weighted cohort, not any single conformer, including the crystal structure, can account for the ensemble behaviors observed by small-angle X-ray scattering (SAXS). All conformers except one are functionally active in terms of ligand binding. Our findings provide direct visual evidence that the sequence-structure relationship of RNA under physiologically relevant solution conditions is more complex than the one-to-one relationship for well-structured proteins. The direct visualization of conformational and architectural ensembles at the single-molecule level in solution may suggest new approaches to RNA structural analyses. RNA conformational heterogeneity is important to diverse functions. Here, the authors use AFM to directly visualize individual RNA molecules that are in various conformational states under near physiological solution conditions for the first time.

T-box riboswitches are multi-domain noncoding RNAs that surveil individual amino acid availabilities in most Gram-positive bacteria. T-boxes directly bind specific tRNAs, query their aminoacylation status to detect starvation, and feedback control the transcription or translation of downstream amino-acid metabolic genes. Most T-boxes rapidly recruit their cognate tRNA ligands through an intricate three-way stem I-stem II-tRNA interaction, whose establishment is not understood. Using single-molecule FRET, SAXS, and time-resolved fluorescence, we find that the free T-box RNA assumes a broad distribution of open, semi-open, and closed conformations that only slowly interconvert. tRNA directly binds all three conformers with distinct kinetics, triggers nearly instantaneous collapses of the open conformations, and returns the T-box RNA to their pre-binding conformations upon dissociation. This scissors-like dynamic behavior is enabled by a hinge-like pseudoknot domain which poises the T-box for rapid tRNA-induced domain closure. This study reveals tRNA-chaperoned folding of flexible, multi-domain mRNAs through a Venus flytrap-like mechanism. T-box riboswitch RNAs directly bind to specific tRNA and regulate the transcription or translation of downstream genes in bacteria. Using single-molecule FRET and ensemble biophysical analyses, here the authors uncover a Venus flytrap-like mechanism where tRNA binding to a T-box riboswitch mRNA triggers its rapid domain closure.

Much of the human genome is transcribed into RNAs 1 , many of which contain structural elements that are important for their function. Such RNA molecules—including those that are structured and well-folded 2 —are conformationally heterogeneous and flexible, which is a prerequisite for function 3 , 4 , but this limits the applicability of methods such as NMR, crystallography and cryo-electron microscopy for structure elucidation. Moreover, owing to the lack of a large RNA structure database, and no clear correlation between sequence and structure, approaches such as AlphaFold 5 for protein structure prediction do not apply to RNA. Therefore, determining the structures of heterogeneous RNAs remains an unmet challenge. Here we report holistic RNA structure determination method using atomic force microscopy, unsupervised machine learning and deep neural networks (HORNET), a novel method for determining three-dimensional topological structures of RNA using atomic force microscopy images of individual molecules in solution. Owing to the high signal-to-noise ratio of atomic force microscopy, this method is ideal for capturing structures of large RNA molecules in distinct conformations. In addition to six benchmark cases, we demonstrate the utility of HORNET by determining multiple heterogeneous structures of RNase P RNA and the HIV-1 Rev response element (RRE) RNA. Thus, our method addresses one of the major challenges in determining heterogeneous structures of large and flexible RNA molecules, and contributes to the fundamental understanding of RNA structural biology. HORNET, a method that uses unsupervised machine learning and deep neural networks to analyse atomic force microscopy data enables structural determination of RNA molecules in multiple conformations.

Long non-coding RNAs (lncRNAs) play pivotal roles in diverse cellular processes ranging from gene regulation and chromatin remodeling to RNA stability and epigenetic modifications. Despite the identification of approximately 95,000 lncRNA genes in humans, our understanding of their structure–function relationships remains very limited. This review examines the current state of lncRNA structure determination. We briefly discuss the advantages and limitations of experimental approaches—including chemical probing methods such as SHAPE and DMS—as well as the challenges inherent to computational predictions, particularly given RNA's dynamic nature, structural heterogeneity, and the energy degeneracy of its building blocks. The review also highlights the difficulties in predicting long-range interactions, including pseudoknots, which are essential for global folding of large RNAs, and discusses how elevated, nonphysiologically Mg 2 ⁺ concentrations used in many experiments can distort our perception of native RNA conformations. Recent advances in cryo-electron microscopy and atomic force microscopy, coupled with machine learning algorithms, offer promising strategies to capture the realistic conformational landscapes of RNAs, including lncRNAs, under near-physiological conditions. These advances have the potential to redefine our understanding of lncRNA architectures, their structural dynamics, and how they influence cellular functions, ultimately informing future directions of lncRNA research and opening new frontiers such as structure-based drug discovery and therapeutic interventions targeting lncRNAs.

The cell is constructed by higher-order structures and organelles through complex interactions among distinct structural constituents. The centrosome is a membraneless organelle composed of two microtubule-derived structures called centrioles and an amorphous mass of pericentriolar material. Super-resolution microscopic analyses in various organisms revealed that diverse pericentriolar material proteins are concentrically localized around a centriole in a highly organized manner. However, the molecular nature underlying these organizations remains unknown. Here we show that two human pericentriolar material scaffolds, Cep63 and Cep152, cooperatively generate a heterotetrameric α-helical bundle that functions in conjunction with its neighboring hydrophobic motifs to self-assemble into a higher-order cylindrical architecture capable of recruiting downstream components, including Plk4, a key regulator for centriole duplication. Mutations disrupting the self-assembly abrogate Plk4-mediated centriole duplication. Because pericentriolar material organization is evolutionarily conserved, this work may offer a paradigm for investigating the assembly and function of centrosomal scaffolds in various organisms. The centrosome is a membraneless organelle composed of two centrioles and an amorphous pericentriolar material but the overall centrosome organizations remains unknown. Here authors show that two scaffold proteins, Cep63 and Cep152, self-assemble into a higher-order cylindrical architecture capable of recruiting downstream components, including Plk4.

Site-specific incorporation of labeled nucleotides is an extremely useful synthetic tool for many structural studies (e.g., NMR, electron paramagnetic resonance (EPR), fluorescence resonance energy transfer (FRET), and X-ray crystallography) of RNA. However, specific-position-labeled RNAs >60 nt are not commercially available on a milligram scale. Position-selective labeling of RNA (PLOR) has been applied to prepare large RNAs labeled at desired positions, and all the required reagents are commercially available. Here, we present a step-by-step protocol for the solid-liquid hybrid phase method PLOR to synthesize 71-nt RNA samples with three different modification applications, containing (i) a 13 C15 N-labeled segment; (ii) discrete residues modified with Cy3, Cy5, or biotin; or (iii) two iodo-U residues. The flexible procedure enables a wide range of downstream biophysical analyses using precisely localized functionalized nucleotides. All three RNAs were obtained in <2 d, excluding time for preparing reagents and optimizing experimental conditions. With optimization, the protocol can be applied to other RNAs with various labeling schemes, such as ligation of segmentally labeled fragments.

Due to the paucity of known RNA structures, experimental phasing is crucial for obtaining three-dimensional structures of RNAs by X-ray crystallography. Covalent attachment of heavy atoms to RNAs is one of the most useful strategies to facilitate phase determination. However, this approach is limited by the inefficiency or inability to synthesize large RNAs (>60 nucleotides) site-specifically labeled with heavy atoms using traditional methods. Here, we applied our recently reported method, PLOR (position-selective labeling of RNA) to incorporate 5-iodouridine at specific positions in the adenine riboswitch RNA aptamer domain, which was then used for crystallization and subsequent de novo SAD phasing. PLOR is a powerful tool to improve the efficiency of obtaining RNA structures de novo by X-ray crystallography.

Biomaterials can improve cancer immunotherapies by controlling their release and thereby optimizing their time‐dependent engagement of the immune system. In this study, an approach is described to control the release of a potent immunostimulant—CpG oligodeoxynucleotide—from a genetically‐encoded elastin‐like polypeptide (ELP) depot. A CpG‐binding ELP containing an oligolysine domain (ELP‐Lys12) is synthesized that electrostatically complexes CpG and formulate it with an excipient ELP. The ELP‐CpG complex retains the thermally responsive phase behavior of the parent ELP, transitioning into a viscous depot at body temperature. Stepwise addition of excipient ELP predictably changes ELP‐CpG transition temperature, depot dissolution kinetics, and retention of CpG within the depot. Mixtures of ELP‐Lys12, excipient ELP, and CpG undergo microphase separation, forming a porous, sponge‐like depot that contains tunable amounts of soluble CpG in the pores. In vivo, the modified formulations exhibit varying degrees of CpG retention over multiple weeks following a single intratumoral injection. Finally, by modifying the release kinetics of CpG, optimized ELP‐CpG achieves greater reduction of metastatic disease in a murine metastatic breast cancer model than soluble CpG. These results demonstrate that ELPs can be used to precisely tune the release kinetics of immunotherapies for better outcomes in the treatment of metastatic cancer. We show that intratumoral release of an immunotherapy—CpG —can be tuned with molecular precision by complexing it to an elastin‐like polypeptide (ELP). Injecting ELP‐CpG and an excipient ELP into tumors drives thermally‐triggered phase separation into a micro‐scale “sponge” depot that releases CpG at rates depending on the ELP composition, which correlates with control of metastatic breast cancer in mice.