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Proceedings of the National Academy of... May 2022The transcriptome represents an attractive but underused set of targets for small-molecule ligands. Here, we devise a technology that leverages fragment-based screening...
The transcriptome represents an attractive but underused set of targets for small-molecule ligands. Here, we devise a technology that leverages fragment-based screening and SHAPE-MaP RNA structure probing to discover small-molecule fragments that bind an RNA structure of interest. We identified fragments and cooperatively binding fragment pairs that bind to the thiamine pyrophosphate (TPP) riboswitch with millimolar to micromolar affinities. We then used structure-activity relationship information to efficiently design a linked-fragment ligand, with no resemblance to the native ligand, with high ligand efficiency and druglikeness, that binds to the TPP thiM riboswitch with high nanomolar affinity and that modulates RNA conformation during cotranscriptional folding. Principles from this work are broadly applicable, leveraging cooperativity and multisite binding, for developing high-quality ligands for diverse RNA targets.
Topics: Base Pairing; Ligands; RNA Folding; Riboswitch; Small Molecule Libraries; Structure-Activity Relationship; Thiamine Pyrophosphate; Transcription, Genetic
PubMed: 35561226
DOI: 10.1073/pnas.2122660119 -
Nature Chemistry Jun 2021RNA origami is a framework for the modular design of nanoscaffolds that can be folded from a single strand of RNA and used to organize molecular components with...
RNA origami is a framework for the modular design of nanoscaffolds that can be folded from a single strand of RNA and used to organize molecular components with nanoscale precision. The design of genetically expressible RNA origami, which must fold cotranscriptionally, requires modelling and design tools that simultaneously consider thermodynamics, the folding pathway, sequence constraints and pseudoknot optimization. Here, we describe RNA Origami Automated Design software (ROAD), which builds origami models from a library of structural modules, identifies potential folding barriers and designs optimized sequences. Using ROAD, we extend the scale and functional diversity of RNA scaffolds, creating 32 designs of up to 2,360 nucleotides, five that scaffold two proteins, and seven that scaffold two small molecules at precise distances. Micrographic and chromatographic comparisons of optimized and non-optimized structures validate that our principles for strand routing and sequence design substantially improve yield. By providing efficient design of RNA origami, ROAD may simplify the construction of custom RNA scaffolds for nanomedicine and synthetic biology.
Topics: Base Sequence; Microscopy, Electron, Transmission; Nanostructures; Nanotechnology; Protein Biosynthesis; RNA; RNA Folding; Small Molecule Libraries; Software; Synthetic Biology
PubMed: 33972754
DOI: 10.1038/s41557-021-00679-1 -
Briefings in Bioinformatics Mar 2018Computational programs for predicting RNA sequences with desired folding properties have been extensively developed and expanded in the past several years. Given a... (Review)
Review
Computational programs for predicting RNA sequences with desired folding properties have been extensively developed and expanded in the past several years. Given a secondary structure, these programs aim to predict sequences that fold into a target minimum free energy secondary structure, while considering various constraints. This procedure is called inverse RNA folding. Inverse RNA folding has been traditionally used to design optimized RNAs with favorable properties, an application that is expected to grow considerably in the future in light of advances in the expanding new fields of synthetic biology and RNA nanostructures. Moreover, it was recently demonstrated that inverse RNA folding can successfully be used as a valuable preprocessing step in computational detection of novel noncoding RNAs. This review describes the most popular freeware programs that have been developed for such purposes, starting from RNAinverse that was devised when formulating the inverse RNA folding problem. The most recently published ones that consider RNA secondary structure as input are antaRNA, RNAiFold and incaRNAfbinv, each having different features that could be beneficial to specific biological problems in practice. The various programs also use distinct approaches, ranging from ant colony optimization to constraint programming, in addition to adaptive walk, simulated annealing and Boltzmann sampling. This review compares between the various programs and provides a simple description of the various possibilities that would benefit practitioners in selecting the most suitable program. It is geared for specific tasks requiring RNA design based on input secondary structure, with an outlook toward the future of RNA design programs.
Topics: Algorithms; Animals; Computational Biology; Humans; Models, Molecular; Nucleic Acid Conformation; RNA; RNA Folding; Software
PubMed: 28049135
DOI: 10.1093/bib/bbw120 -
RNA Biology 2014RNAs play pivotal roles in the cell, ranging from catalysis (e.g., RNase P), acting as adaptor molecule (tRNA) to regulation (e.g., riboswitches). Precise understanding... (Review)
Review
RNAs play pivotal roles in the cell, ranging from catalysis (e.g., RNase P), acting as adaptor molecule (tRNA) to regulation (e.g., riboswitches). Precise understanding of its three-dimensional structures has given unprecedented insight into the molecular basis for all of these processes. Nevertheless, structural studies on RNA are still limited by the very special nature of this polymer. The most common methods for the determination of 3D RNA structures are NMR and X-ray crystallography. Both methods have their own set of requirements and give different amounts of information about the target RNA. For structural studies, the major bottleneck is usually obtaining large amounts of highly pure and homogeneously folded RNA. Especially for X-ray crystallography it can be necessary to screen a large number of variants to obtain well-ordered single crystals. In this mini-review we give an overview about strategies for the design, in vitro production, and purification of RNA for structural studies.
Topics: Crystallography, X-Ray; Nuclear Magnetic Resonance, Biomolecular; Nucleic Acid Conformation; RNA; RNA Folding; RNA, Catalytic
PubMed: 24667346
DOI: 10.4161/rna.28076 -
Observation of coordinated RNA folding events by systematic cotranscriptional RNA structure probing.Nature Communications Nov 2023RNA begins to fold as it is transcribed by an RNA polymerase. Consequently, RNA folding is constrained by the direction and rate of transcription. Understanding how RNA...
RNA begins to fold as it is transcribed by an RNA polymerase. Consequently, RNA folding is constrained by the direction and rate of transcription. Understanding how RNA folds into secondary and tertiary structures therefore requires methods for determining the structure of cotranscriptional folding intermediates. Cotranscriptional RNA chemical probing methods accomplish this by systematically probing the structure of nascent RNA that is displayed from an RNA polymerase. Here, we describe a concise, high-resolution cotranscriptional RNA chemical probing procedure called variable length Transcription Elongation Complex RNA structure probing (TECprobe-VL). We demonstrate the accuracy and resolution of TECprobe-VL by replicating and extending previous analyses of ZTP and fluoride riboswitch folding and mapping the folding pathway of a ppGpp-sensing riboswitch. In each system, we show that TECprobe-VL identifies coordinated cotranscriptional folding events that mediate transcription antitermination. Our findings establish TECprobe-VL as an accessible method for mapping cotranscriptional RNA folding pathways.
Topics: RNA Folding; RNA; Nucleic Acid Conformation; Riboswitch; Transcription, Genetic; DNA-Directed RNA Polymerases
PubMed: 38030633
DOI: 10.1038/s41467-023-43395-9 -
Cell Nov 2019Ribosome assembly is an efficient but complex and heterogeneous process during which ribosomal proteins assemble on the nascent rRNA during transcription. Understanding...
Ribosome assembly is an efficient but complex and heterogeneous process during which ribosomal proteins assemble on the nascent rRNA during transcription. Understanding how the interplay between nascent RNA folding and protein binding determines the fate of transcripts remains a major challenge. Here, using single-molecule fluorescence microscopy, we follow assembly of the entire 3' domain of the bacterial small ribosomal subunit in real time. We find that co-transcriptional rRNA folding is complicated by the formation of long-range RNA interactions and that r-proteins self-chaperone the rRNA folding process prior to stable incorporation into a ribonucleoprotein (RNP) complex. Assembly is initiated by transient rather than stable protein binding, and the protein-RNA binding dynamics gradually decrease during assembly. This work questions the paradigm of strictly sequential and cooperative ribosome assembly and suggests that transient binding of RNA binding proteins to cellular RNAs could provide a general mechanism to shape nascent RNA folding during RNP assembly.
Topics: Models, Biological; Nucleic Acid Conformation; Protein Binding; RNA Folding; RNA Stability; RNA, Ribosomal; RNA-Binding Proteins; Transcription, Genetic
PubMed: 31761533
DOI: 10.1016/j.cell.2019.10.035 -
Nucleic Acids Research Sep 2023Riboswitches are regulatory elements found in bacterial mRNAs that control downstream gene expression through ligand-induced conformational changes. Here, we used...
Riboswitches are regulatory elements found in bacterial mRNAs that control downstream gene expression through ligand-induced conformational changes. Here, we used single-molecule FRET to map the conformational landscape of the translational SAM/SAH riboswitch and probe how co-transcriptional ligand-induced conformational changes affect its translation regulation function. Riboswitch folding is highly heterogeneous, suggesting a rugged conformational landscape that allows for sampling of the ligand-bound conformation even in the absence of ligand. The addition of ligand shifts the landscape, favoring the ligand-bound conformation. Mutation studies identified a key structural element, the pseudoknot helix, that is crucial for determining ligand-free conformations and their ligand responsiveness. We also investigated ribosomal binding site accessibility under two scenarios: pre-folding and co-transcriptional folding. The regulatory function of the SAM/SAH riboswitch involves kinetically favoring ligand binding, but co-transcriptional folding reduces this preference with a less compact initial conformation that exposes the Shine-Dalgarno sequence and takes min to redistribute to more compact conformations of the pre-folded riboswitch. Such slow equilibration decreases the effective ligand affinity. Overall, our study provides a deeper understanding of the complex folding process and how the riboswitch adapts its folding pattern in response to ligand, modulates ribosome accessibility and the role of co-transcriptional folding in these processes.
Topics: Nucleic Acid Conformation; Riboswitch; RNA Folding; Base Pairing; Ribosomes; Ligands
PubMed: 37522343
DOI: 10.1093/nar/gkad633 -
Wiley Interdisciplinary Reviews. RNA Sep 2017The database of RNA sequences is exploding, but knowledge of energetics, structures, and dynamics lags behind. All-atom computational methods, such as molecular... (Review)
Review
The database of RNA sequences is exploding, but knowledge of energetics, structures, and dynamics lags behind. All-atom computational methods, such as molecular dynamics, hold promise for closing this gap. New algorithms and faster computers have accelerated progress in improving the reliability and accuracy of predictions. Currently, the methods can facilitate refinement of experimentally determined nuclear magnetic resonance and x-ray structures, but are 'unreliable' for predictions based only on sequence. Much remains to be discovered, however, about the many molecular interactions driving RNA folding and the best way to approximate them quantitatively. The large number of parameters required means that a wide variety of experimental results will be required to benchmark force fields and different approaches. As computational methods become more reliable and accessible, they will be used by an increasing number of biologists, much as x-ray crystallography has expanded. Thus, many fundamental physical principles underlying the computational methods are described. This review presents a summary of the current state of molecular dynamics as applied to RNA. It is designed to be helpful to students, postdoctoral fellows, and faculty who are considering or starting computational studies of RNA. WIREs RNA 2017, 8:e1422. doi: 10.1002/wrna.1422.
Topics: Crystallography, X-Ray; Models, Molecular; RNA; RNA Folding; Thermodynamics
PubMed: 28815951
DOI: 10.1002/wrna.1422 -
Molecular Cell Feb 2021The series of RNA folding events that occur during transcription can critically influence cellular RNA function. Here, we present reconstructing RNA dynamics from data...
The series of RNA folding events that occur during transcription can critically influence cellular RNA function. Here, we present reconstructing RNA dynamics from data (R2D2), a method to uncover details of cotranscriptional RNA folding. We model the folding of the Escherichia coli signal recognition particle (SRP) RNA and show that it requires specific local structural fluctuations within a key hairpin to engender efficient cotranscriptional conformational rearrangement into the functional structure. All-atom molecular dynamics simulations suggest that this rearrangement proceeds through an internal toehold-mediated strand-displacement mechanism, which can be disrupted with a point mutation that limits local structural fluctuations and rescued with compensating mutations that restore these fluctuations. Moreover, a cotranscriptional folding intermediate could be cleaved in vitro by recombinant E. coli RNase P, suggesting potential cotranscriptional processing. These results from experiment-guided multi-scale modeling demonstrate that even an RNA with a simple functional structure can undergo complex folding and processing during synthesis.
Topics: Escherichia coli; Escherichia coli Proteins; RNA Folding; RNA, Bacterial; Ribonuclease P; Signal Recognition Particle
PubMed: 33453165
DOI: 10.1016/j.molcel.2020.12.017 -
Biophysical Journal Oct 2020The ability to accurately predict RNA hairpin structure and stability for different loop sequences and salt conditions is important for understanding, modeling, and...
The ability to accurately predict RNA hairpin structure and stability for different loop sequences and salt conditions is important for understanding, modeling, and designing larger RNA folds. However, traditional RNA secondary structure models cannot treat loop-sequence and ionic effects on RNA hairpin folding. Here, we describe a general, three-dimensional (3D) conformation-based computational method for modeling salt concentration-dependent conformational distributions and the detailed 3D structures for a set of three RNA hairpins that contain a variable, 15-nucleotide loop sequence. For a given RNA sequence, the new, to our knowledge, method integrates a Vfold2D two-dimensional structure folding model with IsRNA coarse-grained molecular dynamics 3D folding simulations and Monte Carlo tightly bound ion estimations of ion-mediated electrostatic interactions. The model predicts free-energy landscapes for the different RNA hairpin-forming sequences with variable salt conditions. The theoretically predicted results agree with the experimental fluorescence measurements, validating the strategy. Furthermore, the theoretical model goes beyond the experimental results by enabling in-depth 3D structural analysis, revealing energetic mechanisms for the sequence- and salt-dependent folding stability. Although the computational framework presented here is developed for RNA hairpin systems, the general method may be applied to investigate other RNA systems, such as multiway junctions or pseudoknots in mixed metal ion solutions.
Topics: Molecular Dynamics Simulation; Nucleic Acid Conformation; RNA; RNA Folding; RNA Stability; Thermodynamics
PubMed: 32949490
DOI: 10.1016/j.bpj.2020.07.042