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RNA Biology Jan 2023An increased appreciation of the role of RNA dynamics in governing RNA function is ushering in a new wave of dynamic RNA synthetic biology. Here, we review recent... (Review)
Review
An increased appreciation of the role of RNA dynamics in governing RNA function is ushering in a new wave of dynamic RNA synthetic biology. Here, we review recent advances in engineering dynamic RNA systems across the molecular, circuit and cellular scales for important societal-scale applications in environmental and human health, and bioproduction. For each scale, we introduce the core concepts of dynamic RNA folding and function at that scale, and then discuss technologies incorporating these concepts, covering new approaches to engineering riboswitches, ribozymes, RNA origami, RNA strand displacement circuits, biomaterials, biomolecular condensates, extracellular vesicles and synthetic cells. Considering the dynamic nature of RNA within the engineering design process promises to spark the next wave of innovation that will expand the scope and impact of RNA biotechnologies.
Topics: Humans; RNA; Synthetic Biology; RNA, Catalytic; Biotechnology; RNA Folding
PubMed: 38044595
DOI: 10.1080/15476286.2023.2269508 -
Proceedings of the National Academy of... Oct 2021Rotavirus genomes are distributed between 11 distinct RNA molecules, all of which must be selectively copackaged during virus assembly. This likely occurs through...
Rotavirus genomes are distributed between 11 distinct RNA molecules, all of which must be selectively copackaged during virus assembly. This likely occurs through sequence-specific RNA interactions facilitated by the RNA chaperone NSP2. Here, we report that NSP2 autoregulates its chaperone activity through its C-terminal region (CTR) that promotes RNA-RNA interactions by limiting its helix-unwinding activity. Unexpectedly, structural proteomics data revealed that the CTR does not directly interact with RNA, while accelerating RNA release from NSP2. Cryo-electron microscopy reconstructions of an NSP2-RNA complex reveal a highly conserved acidic patch on the CTR, which is poised toward the bound RNA. Virus replication was abrogated by charge-disrupting mutations within the acidic patch but completely restored by charge-preserving mutations. Mechanistic similarities between NSP2 and the unrelated bacterial RNA chaperone Hfq suggest that accelerating RNA dissociation while promoting intermolecular RNA interactions may be a widespread strategy of RNA chaperone recycling.
Topics: Cryoelectron Microscopy; Genome, Viral; Models, Molecular; Molecular Chaperones; RNA Folding; RNA, Viral; RNA-Binding Proteins; Ribonucleoproteins; Rotavirus; Viral Genome Packaging; Viral Nonstructural Proteins
PubMed: 34615715
DOI: 10.1073/pnas.2100198118 -
Proceedings of the National Academy of... Oct 2019RNA molecules cannot fold in the absence of counterions. Experiments are typically performed in the presence of monovalent and divalent cations. How to treat the impact...
RNA molecules cannot fold in the absence of counterions. Experiments are typically performed in the presence of monovalent and divalent cations. How to treat the impact of a solution containing a mixture of both ion types on RNA folding has remained a challenging problem for decades. By exploiting the large concentration difference between divalent and monovalent ions used in experiments, we develop a theory based on the reference interaction site model (RISM), which allows us to treat divalent cations explicitly while keeping the implicit screening effect due to monovalent ions. Our theory captures both the inner shell and outer shell coordination of divalent cations to phosphate groups, which we demonstrate is crucial for an accurate calculation of RNA folding thermodynamics. The RISM theory for ion-phosphate interactions when combined with simulations based on a transferable coarse-grained model allows us to predict accurately the folding of several RNA molecules in a mixture containing monovalent and divalent ions. The calculated folding free energies and ion-preferential coefficients for RNA molecules (pseudoknots, a fragment of the rRNA, and the aptamer domain of the adenine riboswitch) are in excellent agreement with experiments over a wide range of monovalent and divalent ion concentrations. Because the theory is general, it can be readily used to investigate ion and sequence effects on DNA properties.
Topics: Cations, Divalent; Cations, Monovalent; Ions; RNA; RNA Folding; Thermodynamics
PubMed: 31570624
DOI: 10.1073/pnas.1911632116 -
Proceedings of the National Academy of... Jan 2022Accurate knowledge of RNA hybridization is essential for understanding RNA structure and function. Here we mechanically unzip and rezip a 2-kbp RNA hairpin and derive...
Accurate knowledge of RNA hybridization is essential for understanding RNA structure and function. Here we mechanically unzip and rezip a 2-kbp RNA hairpin and derive the 10 nearest-neighbor base pair (NNBP) RNA free energies in sodium and magnesium with 0.1 kcal/mol precision using optical tweezers. Notably, force-distance curves (FDCs) exhibit strong irreversible effects with hysteresis and several intermediates, precluding the extraction of the NNBP energies with currently available methods. The combination of a suitable RNA synthesis with a tailored pulling protocol allowed us to obtain the fully reversible FDCs necessary to derive the NNBP energies. We demonstrate the equivalence of sodium and magnesium free-energy salt corrections at the level of individual NNBP. To characterize the irreversibility of the unzipping-rezipping process, we introduce a barrier energy landscape of the stem-loop structures forming along the complementary strands, which compete against the formation of the native hairpin. This landscape correlates with the hysteresis observed along the FDCs. RNA sequence analysis shows that base stacking and base pairing stabilize the stem-loops that kinetically trap the long-lived intermediates observed in the FDC. Stem-loops formation appears as a general mechanism to explain a wide range of behaviors observed in RNA folding.
Topics: Biomechanical Phenomena; Magnesium; Nucleic Acid Conformation; RNA; RNA Folding; Sodium; Thermodynamics
PubMed: 35022230
DOI: 10.1073/pnas.2025575119 -
Bioinformatics (Oxford, England) Sep 2023Understanding RNA folding at the level of secondary structures can give important insights concerning the function of a molecule. We are interested to learn how...
MOTIVATION
Understanding RNA folding at the level of secondary structures can give important insights concerning the function of a molecule. We are interested to learn how secondary structures change dynamically during transcription, as well as whether particular secondary structures form already during or only after transcription. While different approaches exist to simulate cotranscriptional folding, the current strategies for visualization are lagging behind. New, more suitable approaches are necessary to help with exploring the generated data from cotranscriptional folding simulations.
RESULTS
We present DrForna, an interactive visualization app for viewing the time course of a cotranscriptional RNA folding simulation. Specifically, users can scroll along the time axis and see the population of structures that are present at any particular time point.
AVAILABILITY AND IMPLEMENTATION
DrForna is a JavaScript project available on Github at https://github.com/ViennaRNA/drforna and deployed at https://viennarna.github.io/drforna.
Topics: Computer Simulation; RNA Folding
PubMed: 37682108
DOI: 10.1093/bioinformatics/btad555 -
BMC Bioinformatics Dec 2019A pairings of nucleotide sequences. Given this forbidding free-energy landscape, mechanisms have evolved that contribute to a directed and efficient folding process,...
BACKGROUND
A pairings of nucleotide sequences. Given this forbidding free-energy landscape, mechanisms have evolved that contribute to a directed and efficient folding process, including catalytic proteins and error-detecting chaperones. Among structural RNA molecules we make a distinction between "bound" molecules, which are active as part of ribonucleoprotein (RNP) complexes, and "unbound," with physiological functions performed without necessarily being bound in RNP complexes. We hypothesized that unbound molecules, lacking the partnering structure of a protein, would be more vulnerable than bound molecules to kinetic traps that compete with native stem structures. We defined an "ambiguity index"-a normalized function of the primary and secondary structure of an individual molecule that measures the number of kinetic traps available to nucleotide sequences that are paired in the native structure, presuming that unbound molecules would have lower indexes. The ambiguity index depends on the purported secondary structure, and was computed under both the comparative ("gold standard") and an equilibrium-based prediction which approximates the minimum free energy (MFE) structure. Arguing that kinetically accessible metastable structures might be more biologically relevant than thermodynamic equilibrium structures, we also hypothesized that MFE-derived ambiguities would be less effective in separating bound and unbound molecules.
RESULTS
We have introduced an intuitive and easily computed function of primary and secondary structures that measures the availability of complementary sequences that could disrupt the formation of native stems on a given molecule-an ambiguity index. Using comparative secondary structures, the ambiguity index is systematically smaller among unbound than bound molecules, as expected. Furthermore, the effect is lost when the presumably more accurate comparative structure is replaced instead by the MFE structure.
CONCLUSIONS
A statistical analysis of the relationship between the primary and secondary structures of non-coding RNA molecules suggests that stem-disrupting kinetic traps are substantially less prevalent in molecules not participating in RNP complexes. In that this distinction is apparent under the comparative but not the MFE secondary structure, the results highlight a possible deficiency in structure predictions when based upon assumptions of thermodynamic equilibrium.
Topics: Base Pairing; Base Sequence; Calibration; Kinetics; Nucleic Acid Conformation; RNA; RNA Folding; ROC Curve; Thermodynamics
PubMed: 31830902
DOI: 10.1186/s12859-019-3303-6 -
Bioinformatics (Oxford, England) Jan 2016Chemical mapping experiments allow for nucleotide resolution assessment of RNA structure. We demonstrate that different strategies of integrating probing data with...
SUMMARY
Chemical mapping experiments allow for nucleotide resolution assessment of RNA structure. We demonstrate that different strategies of integrating probing data with thermodynamics-based RNA secondary structure prediction algorithms can be implemented by means of soft constraints. This amounts to incorporating suitable pseudo-energies into the standard energy model for RNA secondary structures. As a showcase application for this new feature of the ViennaRNA Package we compare three distinct, previously published strategies to utilize SHAPE reactivities for structure prediction. The new tool is benchmarked on a set of RNAs with known reference structure.
AVAILABILITY AND IMPLEMENTATION
The capability for SHAPE directed RNA folding is part of the upcoming release of the ViennaRNA Package 2.2, for which a preliminary release is already freely available at http://www.tbi.univie.ac.at/RNA.
CONTACT
SUPPLEMENTARY INFORMATION
Supplementary data are available at Bioinformatics online.
Topics: Algorithms; Base Sequence; Escherichia coli; Molecular Sequence Data; Nucleic Acid Conformation; RNA Folding; RNA, Ribosomal; Thermodynamics
PubMed: 26353838
DOI: 10.1093/bioinformatics/btv523 -
Biochemistry Aug 2021RNA regulates myriad cellular events such as transcription, translation, and splicing. To perform these essential functions, RNA often folds into complex tertiary...
RNA regulates myriad cellular events such as transcription, translation, and splicing. To perform these essential functions, RNA often folds into complex tertiary structures in which its negatively charged ribose-phosphate backbone interacts with metal ions. Magnesium, the most abundant divalent metal ion in cells, neutralizes the backbone, thereby playing essential roles in RNA folding and function. This has been known for more than 50 years, and there are now thousands of studies, most of which have used ≥10 mM free Mg ions to achieve optimal RNA folding and function. In the cell, however, concentrations of free Mg ions are much lower, with most Mg ions chelated by metabolites. In this Perspective, we curate data from a number of sources to provide extensive summaries of cellular concentrations of metabolites that bind Mg and to estimate cellular concentrations of metabolite-chelated Mg species, in the representative prokaryotic and eukaryotic systems , , and iBMK cells. Recent research from our lab and others has uncovered the fact that such weakly chelated Mg ions can enhance RNA function, including its thermodynamic stability, chemical stability, and catalysis. We also discuss how metabolite-chelated Mg complexes may have played roles in the origins of life. It is clear from this analysis that bound Mg should not be simply considered non-RNA-interacting and that future RNA research, as well as protein research, could benefit from considering chelated magnesium.
Topics: Animals; Biocatalysis; Cations, Divalent; Cell Line; Escherichia coli; Magnesium; Metabolome; Mice; RNA; RNA Folding; Saccharomyces cerevisiae
PubMed: 34319696
DOI: 10.1021/acs.biochem.1c00012 -
Wiley Interdisciplinary Reviews. RNA 2014RNA hairpins are the most commonly occurring secondary structural elements in RNAs and serve as nucleation sites for RNA folding, RNA-RNA, and RNA-protein interactions.... (Review)
Review
RNA hairpins are the most commonly occurring secondary structural elements in RNAs and serve as nucleation sites for RNA folding, RNA-RNA, and RNA-protein interactions. RNA hairpins are frequently capped by tetraloops, and based on sequence similarity, three broad classes of RNA tetraloops have been defined: GNRA, UNCG, and CUYG. Other classes such as the UYUN tetraloop in histone mRNAs, the UGAA in 16S rRNA, the AUUA tetraloop from the MS2 bacteriophage, and the AGNN tetraloop that binds RNase III have also been characterized. The tetraloop structure is compact and is usually characterized by a paired interaction between the first and fourth nucleotides. The two unpaired nucleotides in the loop are usually involved in base-stacking or base-phosphate hydrogen bonding interactions. Several structures of RNA tetraloops, free and complexed to other RNAs or proteins, are now available and these studies have increased our understanding of the diverse mechanisms by which this motif is recognized. RNA tetraloops can mediate RNA-RNA contacts via the tetraloop-receptor motif, kissing hairpin loops, A-minor interactions, and pseudoknots. While these RNA-RNA interactions are fairly well understood, how RNA-binding proteins recognize RNA tetraloops and tetraloop-like motifs remains unclear. In this review, we summarize the structures of RNA tetraloop-protein complexes and the general themes that have emerged on sequence- and structure-specific recognition of RNA tetraloops. We highlight how proteins achieve molecular recognition of this nucleic acid motif, the structural adaptations observed in the tetraloop to accommodate the protein-binding partner, and the role of dynamics in recognition.
Topics: Animals; Binding Sites; Humans; Models, Molecular; Nucleic Acid Conformation; Protein Structure, Secondary; RNA; RNA Folding; RNA-Binding Proteins
PubMed: 24124096
DOI: 10.1002/wrna.1196 -
Science (New York, N.Y.) Dec 2017Self-folding of an information-carrying polymer into a defined structure is foundational to biology and offers attractive potential as a synthetic strategy. Although...
Self-folding of an information-carrying polymer into a defined structure is foundational to biology and offers attractive potential as a synthetic strategy. Although multicomponent self-assembly has produced complex synthetic nanostructures, unimolecular folding has seen limited progress. We describe a framework to design and synthesize a single DNA or RNA strand to self-fold into a complex yet unknotted structure that approximates an arbitrary user-prescribed shape. We experimentally construct diverse multikilobase single-stranded structures, including a ~10,000-nucleotide (nt) DNA structure and a ~6000-nt RNA structure. We demonstrate facile replication of the strand in vitro and in living cells. The work here thus establishes unimolecular folding as a general strategy for constructing complex and replicable nucleic acid nanostructures, and expands the design space and material scalability for bottom-up nanotechnology.
Topics: DNA Replication; DNA, Single-Stranded; Escherichia coli; Nanostructures; Nanotechnology; Nucleotides; RNA; RNA Folding
PubMed: 29242318
DOI: 10.1126/science.aao2648