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1.
Nucleic Acids Res ; 51(1): 54-67, 2023 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-36610789

RESUMO

Riboswitches are conserved non-coding domains in bacterial mRNA with gene regulation function that are essential for maintaining enzyme co-factor metabolism. Recently, the pnuC RNA motif was reported to selectively bind nicotinamide adenine dinucleotide (NAD+), defining a novel class of NAD+ riboswitches (NAD+-II) according to phylogenetic analysis. To reveal the three-dimensional architecture and the ligand-binding mode of this riboswitch, we solved the crystal structure of NAD+-II riboswitch in complex with NAD+. Strikingly and in contrast to class-I riboswitches that form a tight recognition pocket for the adenosine diphosphate (ADP) moiety of NAD+, the class-II riboswitches form a binding pocket for the nicotinamide mononucleotide (NMN) portion of NAD+ and display only unspecific interactions with the adenosine. We support this finding by an additional structure of the class-II RNA in complex with NMN alone. The structures define a novel RNA tertiary fold that was further confirmed by mutational analysis in combination with isothermal titration calorimetry (ITC), and 2-aminopurine-based fluorescence spectroscopic folding studies. Furthermore, we truncated the pnuC RNA motif to a short RNA helical scaffold with binding affinity comparable to the wild-type motif to allude to the potential of engineering the NAD+-II motif for biotechnological applications.


Assuntos
Riboswitch , NAD/metabolismo , Filogenia , Ligantes , RNA/genética
2.
Methods Mol Biol ; 2570: 155-173, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36156781

RESUMO

Fluorogenic RNA aptamers are synthetic RNAs that have been evolved by in vitro selection methods to bind and light up conditionally fluorescent organic ligands. Compared with other probes for RNA detection, they are less invasive than hybridization-based methods (FISH, molecular beacons) and are considerably smaller than fluorescent protein-recruiting systems (MS2, Pumilio variants). Fluorogenic aptamers have therefore found widespread use as genetically encodable tags for RNA detection in live cells and have also been used in combination with riboswitches to construct versatile metabolite sensors for in vitro use. Their success builds on a fundamental understanding of their three-dimensional structure to explain the mechanisms of ligand interaction and to rationally design functional aptamer devices. In this protocol, we describe a supramolecular FRET-based structure probing method for fluorogenic aptamers that exploits distance- and orientation-dependent energy transfer efficiencies between site-specifically incorporated fluorescent nucleoside analogs and non-covalently bound ligands, exemplified by 4-cyanoindol riboside (4CI) and the DMHBI+-binding RNA aptamer Chili. This method yields structural restraints that bridge the gap between traditional low-resolution secondary structure probing methods and more elaborate high-resolution methods such as X-ray crystallography and NMR spectroscopy.


Assuntos
Aptâmeros de Nucleotídeos , Riboswitch , Aptâmeros de Nucleotídeos/química , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/química , Ligantes , Nucleosídeos , RNA/genética
3.
Methods Mol Biol ; 2570: 63-71, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36156774

RESUMO

SELEX has enabled the selection of aptamers, nucleic acids that can bind a defined ligand, in some cases with exceptionally high affinity and specificity. The SELEX protocol has been adapted many times to fit a variety of needs. This protocol describes such an adaptation, namely, RNA-Capture SELEX that we have used to successfully develop small molecule-binding RNA aptamers. Our proposed method specifically selects not only for excellent binding but also for conformational switching. In consequence, we found this SELEX method to be particularly suitable for identifying aptamers for further application in synthetic riboswitch engineering.


Assuntos
Aptâmeros de Nucleotídeos , Riboswitch , Aptâmeros de Nucleotídeos/química , Ligantes , Fenômenos Magnéticos , RNA , Técnica de Seleção de Aptâmeros/métodos , Estreptavidina/metabolismo
4.
Compr Rev Food Sci Food Saf ; 22(1): 451-472, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36511082

RESUMO

Food safety has always been a hot issue of social concern, and biosensing has been widely used in the field of food safety detection. Compared with traditional aptamer-based biosensors, aptamer-based riboswitch biosensing represents higher precision and programmability. A riboswitch is an elegant example of controlling gene expression, where the target is coupled to the aptamer domain, resulting in a conformational change in the downstream expression domain and determining the signal output. Riboswitch-based biosensing can be extensively applied to the portable real-time detection of food samples. The numerous key features of riboswitch-based biosensing emphasize their sustainability, renewable, and testing, which promises to transform engineering applications in the field of food safety. This review covers recent developments in riboswitch-based biosensors. The brief history, definition, and modular design (regulatory mode, reporter, and expression platform) of riboswitch-based biosensors are explained for better insight into the design and construction. We summarize recent advances in various riboswitch-based biosensors involving theophylline, malachite green, tetracycline, neomycin, fluoride, thrombin, naringenin, ciprofloxacin, and paromomycin, aiming to provide general guidance for the design of riboswitch-based biosensors. Finally, the challenges and prospects are also summarized as a way forward stratagem and signs of progress.


Assuntos
Técnicas Biossensoriais , Riboswitch , Técnicas Biossensoriais/métodos , Antibacterianos
5.
RNA Biol ; 20(1): 10-19, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36548032

RESUMO

Riboswitches are regulatory RNAs that specifically bind a small molecule or ion. Like metabolite-binding proteins, riboswitches can evolve new ligand specificities, and some examples of this phenomenon have been validated. As part of work based on comparative genomics to discover novel riboswitches, we encountered a candidate riboswitch with striking similarities to the recently identified guanidine-IV riboswitch. This candidate riboswitch, the Gd4v motif, is predicted in four distinct bacterial phyla, thus almost as widespread as the guanidine-IV riboswitch. Bioinformatic and experimental analysis suggest that the Gd4v motif is a riboswitch that binds a ligand other than guanidine. It is found associated with gene classes that differ from genes regulated by confirmed guanidine riboswitches. In inline-probing assays, we showed that free guanidine binds only weakly to one of the tested sequences of the variant. Further tested compounds did not show binding, attenuation of transcription termination, or activation of a genetic reporter construct. We characterized an N-acetyltransferase frequently associated with the Gd4v motif and compared its substrate preference to an N-acetyltransferase that occurs under control of guanidine-IV riboswitches. The substrates of this Gd4v-motif-associated enzyme did not show activity for Gd4v RNA binding or transcription termination. Hence, the ligand of the candidate riboswitch motif remains unidentified. The variant RNA motif is predominantly found in gut metagenome sequences, hinting at a ligand that is highly relevant in this environment. This finding is a first step to determining the identity of this unknown ligand, and understanding how guanidine-IV-riboswitch-like structures can evolve to bind different ligands.


Assuntos
Riboswitch , Guanidina/química , Guanidina/metabolismo , Conformação de Ácido Nucleico , Ligantes , Guanidinas/metabolismo , Acetiltransferases/genética , Acetiltransferases/metabolismo
6.
ACS Synth Biol ; 12(1): 35-42, 2023 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-36566430

RESUMO

An RNA aptamer that induces suitable conformational changes upon binding to a user-defined ligand allows us to artificially construct a riboswitch, a ligand-dependent and cis-acting gene regulatory RNA. Although such an aptamer can be obtained through in vitro selection, it is still challenging to rationally expand the variety of orthogonal ligand/aptamer (ligand/riboswitch) pairs. To achieve this in a facile, selection-free way, we herein focused on a specific type of ligand, 6-nt nanosized DNA (nDNA) and its aptamer that was previously selected to construct a eukaryotic artificial riboswitch. Specifically, we merely mutated one or more possible Watson-Crick base pairs in the nDNA/aptamer (nDNA/riboswitch) interactions into another base pair or pairs. Using two sets that each had 16 comprehensive mutations, we obtained three groups of several orthogonal nDNA/riboswitch pairs. These pairs could be used to create complex gene circuits, including multiple simultaneous and/or multistep cascading regulations in synthetic biology.


Assuntos
Aptâmeros de Nucleotídeos , Riboswitch , Riboswitch/genética , Ligantes , RNA , Pareamento de Bases/genética , Aptâmeros de Nucleotídeos/metabolismo , Conformação de Ácido Nucleico
7.
Metab Eng ; 75: 143-152, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36549411

RESUMO

Flavonoids are a group of secondary metabolites from plants that have received attention as high value-added pharmacological substances. Recently, a robust and efficient bioprocess using recombinant microbes has emerged as a promising approach to supply flavonoids. In the flavonoid biosynthetic pathway, the rate of chalcone synthesis, the first committed step, is a major bottleneck. However, chalcone synthase (CHS) engineering was difficult because of high-level conservation and the absence of effective screening tools, which are limited to overexpression or homolog-based combinatorial strategies. Furthermore, it is necessary to precisely regulate the metabolic flux for the optimum availability of malonyl-CoA, a substrate of chalcone synthesis. In this study, we engineered CHS and optimized malonyl-CoA availability to establish a platform strain for naringenin production, a key molecular scaffold for various flavonoids. First, we engineered CHS through synthetic riboswitch-based high-throughput screening of rationally designed mutant libraries. Consequently, the catalytic efficiency (kcat/Km) of the optimized CHS enzyme was 62% higher than that of the wild-type enzyme. In addition to CHS engineering, we designed genetic circuits using transcriptional repressors to fine-tune the malonyl-CoA availability. The best mutant with synergistic effects of the engineered CHS and the optimized genetic circuit produced 98.71 mg/L naringenin (12.57 mg naringenin/g glycerol), which is the highest naringenin concentration and yield from glycerol in similar culture conditions reported to date, a 2.5-fold increase compared to the parental strain. Overall, this study provides an effective strategy for efficient production of flavonoids.


Assuntos
Chalconas , Flavanonas , Riboswitch , Flavonoides/genética , Glicerol , Flavanonas/genética , Malonil Coenzima A/genética , Malonil Coenzima A/metabolismo , Engenharia Metabólica
8.
Sci Rep ; 12(1): 19145, 2022 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-36352003

RESUMO

Lithium is rare in Earth's crust compared to the biologically relevant alkali metal cations sodium and potassium but can accumulate to toxic levels in some environments. We report the experimental validation of two distinct bacterial riboswitch classes that selectively activate gene expression in response to elevated Li+ concentrations. These RNAs commonly regulate the expression of nhaA genes coding for ion transporters that weakly discriminate between Na+ and Li+. Our findings demonstrated that the primary function of Li+ riboswitches and associated NhaA transporters is to prevent Li+ toxicity, particularly when bacteria are living at high pH. Additional riboswitch-associated genes revealed how some cells defend against the deleterious effects of Li+ in the biosphere, which might become more problematic as its industrial applications increase.


Assuntos
Riboswitch , Riboswitch/genética , Lítio/farmacologia , Lítio/metabolismo , Genes Bacterianos , Bactérias/genética , Bactérias/metabolismo , Sódio/metabolismo , Cátions/metabolismo , Proteínas de Membrana Transportadoras/metabolismo
9.
J Phys Chem B ; 126(46): 9457-9464, 2022 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-36379020

RESUMO

Mg2+ is well known to facilitate the structural folding of RNA. However, the thermodynamic and dynamic roles of Mg2+ in RNA folding remain elusive. Here, we exploit single-molecule fluorescence resonance energy transfer (smFRET) and isothermal titration calorimetry (ITC) to study the mechanism of Mg2+ in facilitating the folding of thiamine pyrophosphate (TPP) riboswitch RNA. The results of smFRET identify that the presence of Mg2+ compacts the RNA and enlarges the conformational dispersity among individual RNA molecules, resulting in a large gain of entropy. The compact yet flexible conformations triggered by Mg2+ may help the riboswitch recognize its specific ligand and further fold. This is supported by the ITC experiments, in which the Mg2+-induced RNA folding is driven by entropy (ΔS) instead of enthalpy (ΔH). Our results complement the understanding of the Mg2+-induced RNA folding. The strategy developed in this work can be used to model other RNAs' folding under different conditions.


Assuntos
Riboswitch , Tiamina Pirofosfato/química , Tiamina Pirofosfato/genética , Tiamina Pirofosfato/metabolismo , Entropia , Conformação de Ácido Nucleico , Dobramento de RNA , RNA Bacteriano/química
10.
J Phys Chem B ; 126(47): 9781-9789, 2022 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-36399551

RESUMO

Nanoscopic differences in free volume result in pressure-dependent changes in free energies which can therefore impact folding/unfolding stability of biomolecules. Although such effects are typically insignificant under ambient pressure conditions, they are crucially important for deep ocean marine life, where the hydraulic pressure can be on the kilobar scale. In this work, single molecule FRET spectroscopy is used to study the effects of pressure on both the kinetics and overall thermodynamics for folding/unfolding of the manganese riboswitch. Detailed pressure-dependent analysis of the conformational kinetics allows one to extract precision changes (σ ≲ 4-8 Å3) in free volumes not only between the fully folded/unfolded conformations but also with respect to the folding transition state of the manganese riboswitch. This permits first extraction of a novel "reversible work" free energy (PΔV) landscape, which reveals a monotonic increase in manganese riboswitch volume along the folding coordinate. Furthermore, such a tool permits exploration of pressure-dependent effects on both Mn2+ binding and riboswitch folding, which demonstrate that ligand attachment stabilizes the riboswitch under pressure by decreasing the volume increase upon folding (ΔΔV < 0). Such competition between ligand binding and pressure-induced denaturation dynamics could be of significant evolutionary advantage, compensating for a weakening in riboswitch tertiary structure with pressure-mediated ligand binding and promotion of folding response.


Assuntos
Riboswitch , Ligantes , Manganês , Cinética , Íons
11.
Biomolecules ; 12(11)2022 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-36358983

RESUMO

Residual dipolar couplings (RDCs) are increasingly used for high-throughput NMR-based structural studies and to provide long-range angular constraints to validate and refine structures of various molecules determined by X-ray crystallography and NMR spectroscopy. RDCs of a given molecule can be measured in an anisotropic environment that aligns in an external magnetic field. Here, we demonstrate the first application of polymer-based nanodiscs for the measurement of RDCs from nucleic acids. Polymer-based nanodiscs prepared using negatively charged SMA-EA polymer and zwitterionic DMPC lipids were characterized by size-exclusion chromatography, 1H NMR, dynamic light-scattering, and 2H NMR. The magnetically aligned polymer-nanodiscs were used as an alignment medium to measure RDCs from a 13C/15N-labeled fluoride riboswitch aptamer using 2D ARTSY-HSQC NMR experiments. The results showed that the alignment of nanodiscs is stable for nucleic acids and nanodisc-induced RDCs fit well with the previously determined solution structure of the riboswitch. These results demonstrate that SMA-EA-based lipid-nanodiscs can be used as a stable alignment medium for high-resolution structural and dynamical studies of nucleic acids, and they can also be applicable to study various other biomolecules and small molecules in general.


Assuntos
Ácidos Nucleicos , Riboswitch , Ácidos Nucleicos/química , Polímeros/química , Espectroscopia de Ressonância Magnética/métodos , Imageamento por Ressonância Magnética
12.
Genes (Basel) ; 13(11)2022 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-36360167

RESUMO

RNA molecules, in one form or another, are involved in almost all aspects of cell physiology, as well as in disease development. The diversity of the functional roles of RNA comes from its intrinsic ability to adopt complex secondary and tertiary structures, rivaling the diversity of proteins. The RNA molecules form dynamic ensembles of many interconverting conformations at a timescale of seconds, which is a key for understanding how they execute their cellular functions. Given the crucial role of RNAs in various cellular processes, we need to understand the RNA molecules from a structural perspective. Central to this review are studies aimed at revealing the regulatory role of conformational equilibria in RNA in humans to understand genetic diseases such as cancer and neurodegenerative diseases, as well as in pathogens such as bacteria and viruses so as to understand the progression of infectious diseases. Furthermore, we also summarize the prior studies on the use of RNA structures as platforms for the rational design of small molecules for therapeutic applications.


Assuntos
Riboswitch , Humanos , Conformação de Ácido Nucleico , RNA/química , Proteínas/genética
13.
Commun Biol ; 5(1): 1120, 2022 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-36273041

RESUMO

Riboswitches normally regulate gene expression through structural changes in response to the specific binding of cellular metabolites or metal ions. Taking add adenine riboswitch as an example, we explore the influences of metal ions (especially for K+ and Mg2+ ions) on the structure and dynamics of riboswitch aptamer (with and without ligand) by using molecular dynamic (MD) simulations. Our results show that a two-state transition marked by the structural deformation at the connection of J12 and P1 (CJ12-P1) is not only related to the binding of cognate ligands, but also strongly coupled with the change of metal ion environments. Moreover, the deformation of the structure at CJ12-P1 can be transmitted to P1 directly connected to the expression platform in multiple ways, which will affect the structure and stability of P1 to varying degrees, and finally change the regulation state of this riboswitch.


Assuntos
Aptâmeros de Nucleotídeos , Riboswitch , Ligantes , Adenina , Conformação de Ácido Nucleico , Aptâmeros de Nucleotídeos/química , Íons
14.
Chempluschem ; 87(11): e202200256, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36220343

RESUMO

High-resolution mass spectrometry was used for the label-free, direct localization and relative quantification of CMC+ -modifications of a neomycin-sensing riboswitch aptamer domain in the absence and presence of the aminoglycoside ligands neomycin B, ribostamycin, and paromomycin. The chemical probing and MS data for the free riboswitch show high exposure to solvent of the uridine nucleobases U7, U8, U13, U14, U18 as part of the proposed internal and apical loops, but those of U10 and U21 as part of the proposed internal loop were found to be far less exposed than expected. Thus, our data are in better agreement with the proposed secondary structure of the riboswitch in complexes with aminoglycosides than with that of free RNA. For the riboswitch in complexes with neomycin B, ribostamycin, and paromomycin, we found highly similar CMC+ -modification patterns and excellent agreement with previous NMR studies. Differences between the chemical probing and MS data in the absence and presence of the aminoglycoside ligands were quantitative rather than qualitative (i. e., the same nucleobases were labeled, but to different extents) and can be rationalized by stabilization of both the proposed bulge and the apical loop by aminoglycoside binding. Our study shows that chemical probing and mass spectrometry can provide important structural information and complement other techniques such as NMR spectroscopy.


Assuntos
Riboswitch , Neomicina/química , Neomicina/metabolismo , Ribostamicina/química , Ribostamicina/metabolismo , RNA , Paromomicina/química , Paromomicina/metabolismo , Framicetina , Aminoglicosídeos , Antibacterianos , Ligantes , Oligonucleotídeos/química , Espectrometria de Massas
15.
Microbiol Spectr ; 10(5): e0336822, 2022 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-36190429

RESUMO

The trace metal manganese in excess affects iron-sulfur cluster and heme-protein biogenesis, eliciting cellular toxicity. The manganese efflux protein MntP is crucial to evading manganese toxicity in bacteria. Recently, two Mn-sensing riboswitches upstream of mntP and alx in Escherichia coli have been reported to mediate the upregulation of their expression under manganese shock. As the alx riboswitch is also responsive to alkaline shock administered externally, it is intriguing whether the mntP riboswitch is also responsive to alkaline stress. Furthermore, how both manganese and alkaline pH simultaneously regulate these two riboswitches under physiological conditions is a puzzle. Using multiple approaches, we show that manganese shock activated glutamine synthetase (GlnA) and glutaminases (GlsA and GlsB) to spike ammonia production in E. coli. The elevated ammonia intrinsically alkalizes the cytoplasm. We establish that this alkalization under manganese stress is crucial for attaining the highest degree of riboswitch activation. Additional studies showed that alkaline pH promotes a 17- to 22-fold tighter interaction between manganese and the mntP riboswitch element. Our study uncovers a physiological linkage between manganese efflux and pH homeostasis that mediates enhanced manganese tolerance. IMPORTANCE Riboswitch RNAs are cis-acting elements that can adopt alternative conformations in the presence or absence of a specific ligand(s) to modulate transcription termination or translation initiation processes. In the present work, we show that manganese and alkaline pH are both necessary for maximal mntP riboswitch activation to mitigate the manganese toxicity. This study bridges the gap between earlier studies that separately emphasize the importance of alkaline pH and manganese in activating the riboswitches belonging to the yybP-ykoY family. This study also ascribes a physiological relevance as to how manganese can rewire cellular physiology to render cytoplasmic pH alkaline for its homeostasis.


Assuntos
Proteínas de Escherichia coli , Riboswitch , Escherichia coli/genética , Escherichia coli/metabolismo , Manganês/toxicidade , Manganês/química , Manganês/metabolismo , Regulação Bacteriana da Expressão Gênica , Ligantes , Glutamato-Amônia Ligase/metabolismo , Amônia/metabolismo , Ferro/metabolismo , Heme/metabolismo , Enxofre/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Escherichia coli/metabolismo
17.
RNA Biol ; 19(1): 1059-1076, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-36093908

RESUMO

Riboswitch architectures that involve the binding of a single ligand to a single RNA aptamer domain result in ordinary dose-response curves that require approximately a 100-fold change in ligand concentration to cover nearly the full dynamic range for gene regulation. However, by using multiple riboswitches or aptamer domains in tandem, these ligand-sensing structures can produce additional, complex gene control outcomes. In the current study, we have computationally searched for tandem riboswitch architectures in bacteria to provide a more complete understanding of the diverse biological and biochemical functions of gene control elements that are made exclusively of RNA. Numerous different arrangements of tandem homologous riboswitch architectures are exploited by bacteria to create more 'digital' gene control devices, which operate over a narrower ligand concentration range. Also, two heterologous riboswitch aptamers are sometimes employed to create two-input Boolean logic gates with various types of genetic outputs. These findings illustrate the sophisticated genetic decisions that can be made by using molecular sensors and switches based only on RNA.


Assuntos
Aptâmeros de Nucleotídeos , Riboswitch , Aptâmeros de Nucleotídeos/química , Ligantes , RNA , Riboswitch/genética
18.
Proc Natl Acad Sci U S A ; 119(38): e2209608119, 2022 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-36095194

RESUMO

Helicases are ubiquitous motor enzymes that remodel nucleic acids (NA) and NA-protein complexes in key cellular processes. To explore the functional repertoire and specificity landscape of helicases, we devised a screening scheme-Helicase-SELEX (Systematic Evolution of Ligands by EXponential enrichment)-that enzymatically probes substrate and cofactor requirements at global scale. Using the transcription termination Rho helicase of Escherichia coli as a prototype for Helicase-SELEX, we generated a genome-wide map of Rho utilization (Rut) sites. The map reveals many features, including promoter- and intrinsic terminator-associated Rut sites, bidirectional Rut tandems, and cofactor-dependent Rut sites with inverted G > C skewed compositions. We also implemented an H-SELEX variant where we used a model ligand, serotonin, to evolve synthetic Rut sites operating in vitro and in vivo in a ligand-dependent manner. Altogether, our data illustrate the power and flexibility of Helicase-SELEX to seek constitutive or conditional helicase substrates in natural or synthetic NA libraries for fundamental or synthetic biology discovery.


Assuntos
DNA Helicases , Riboswitch , Técnica de Seleção de Aptâmeros , Terminação da Transcrição Genética , Sítios de Ligação , DNA Helicases/química , Escherichia coli/enzimologia , Ligantes , Especificidade por Substrato
19.
Nucleic Acids Res ; 50(20): e120, 2022 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-36166000

RESUMO

RNA molecules can form secondary and tertiary structures that can regulate their localization and function. Using enzymatic or chemical probing together with high-throughput sequencing, secondary structure can be mapped across the entire transcriptome. However, a limiting factor is that only population averages can be obtained since each read is an independent measurement. Although long-read sequencing has recently been used to determine RNA structure, these methods still used aggregate signals across the strands to detect structure. Averaging across the population also means that only limited information about structural heterogeneity across molecules or dependencies within each molecule can be obtained. Here, we present Single-Molecule Structure sequencing (SMS-seq) that combines structural probing with native RNA sequencing to provide non-amplified, structural profiles of individual molecules with novel analysis methods. Our new approach using mutual information enabled single molecule structural interrogation. Each RNA is probed at numerous bases enabling the discovery of dependencies and heterogeneity of structural features. We also show that SMS-seq can capture tertiary interactions, dynamics of riboswitch ligand binding, and mRNA structural features.


Assuntos
Nanoporos , Riboswitch , Análise de Sequência de RNA/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , RNA/genética , RNA/química , Transcriptoma
20.
Int J Biol Macromol ; 222(Pt A): 680-690, 2022 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-36167105

RESUMO

RNA plays a key role in numerous biological processes. Traditional experimental methods have difficulties capturing the structure and dynamic conformation of RNA. Thus, Molecular dynamic simulations (MDs) has become an essential complementary for RNA experiment. However, state-of-the-art RNA force fields have two major limitations of overestimation base stacking propensity and generation of a high ratio of intercalated conformations. Therefore, a two-step strategy was used to optimize the parameters of ff99bsc0χOL3 (named BSFF1) to improve these limitations, which as well adjusted the unbonded parameters of nucleobase heavy atoms and added ζ/α grid-based energy correction map energy term with reweighting. MD simulations of tetranucleotides indicate that BSFF1 can significantly decrease the ratio of intercalated conformations. Tests of single-strand RNA and kink-turn show that BSFF1 force field can reproduce more accurate conformers than ff99bsc0χOL3 force field. BSFF1 can also stabilize the conformers of duplex and riboswitch. The successful ab initio folding of tetraloop further supports the performance of BSFF1. These findings confirm that the newly developed force field BSFF1 can improve the conformer sampling of RNA.


Assuntos
RNA , Riboswitch , RNA/química , Nucleotídeos , Conformação Molecular , Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico
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