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1.
Molecules ; 26(11)2021 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-34205049

RESUMO

Aberrant RNA-protein complexes are formed in a variety of diseases. Identifying the ligands that interfere with their formation is a valuable therapeutic strategy. Molecular simulation, validated against experimental data, has recently emerged as a powerful tool to predict both the pose and energetics of such ligands. Thus, the use of molecular simulation may provide insight into aberrant molecular interactions in diseases and, from a drug design perspective, may allow for the employment of less wet lab resources than traditional in vitro compound screening approaches. With regard to basic research questions, molecular simulation can support the understanding of the exact molecular interaction and binding mode. Here, we focus on examples targeting RNA-protein complexes in neurodegenerative diseases and viral infections. These examples illustrate that the strategy is rather general and could be applied to different pharmacologically relevant approaches. We close this study by outlining one of these approaches, namely the light-controllable association of small molecules with RNA, as an emerging approach in RNA-targeting therapy.


Assuntos
Peptídeos/farmacologia , Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Desenho de Fármacos , Humanos , Ligantes , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Processos Fotoquímicos , Ligação Proteica/efeitos dos fármacos , RNA/química , Proteínas de Ligação a RNA/química
2.
Molecules ; 26(12)2021 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-34200901

RESUMO

Peptides and their synthetic analogs are a class of molecules with enormous relevance as therapeutics for their ability to interact with biomacromolecules like nucleic acids and proteins, potentially interfering with biological pathways often involved in the onset and progression of pathologies of high social impact. Nucleobase-bearing peptides (nucleopeptides) and pseudopeptides (PNAs) offer further interesting possibilities related to their nucleobase-decorated nature for diagnostic and therapeutic applications, thanks to their reported ability to target complementary DNA and RNA strands. In addition, these chimeric compounds are endowed with intriguing self-assembling properties, which are at the heart of their investigation as self-replicating materials in prebiotic chemistry, as well as their application as constituents of innovative drug delivery systems and, more generally, as novel nanomaterials to be employed in biomedicine. Herein we describe the properties of nucleopeptides, PNAs and related supramolecular systems, and summarize some of the most relevant applications of these systems.


Assuntos
Nanoestruturas/química , Ácidos Nucleicos Peptídicos/química , Peptídeos/química , Animais , DNA/química , Humanos , Prebióticos , RNA/química
3.
Nat Commun ; 12(1): 3397, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099665

RESUMO

It is known that an RNA's structure determines its biological function, yet current RNA structure probing methods only capture partial structure information. The ability to measure intact (i.e., full length) RNA structures will facilitate investigations of the functions and regulation mechanisms of small RNAs and identify short fragments of functional sites. Here, we present icSHAPE-MaP, an approach combining in vivo selective 2'-hydroxyl acylation and mutational profiling to probe intact RNA structures. We further showcase the RNA structural landscape of substrates bound by human Dicer based on the combination of RNA immunoprecipitation pull-down and icSHAPE-MaP small RNA structural profiling. We discover distinct structural categories of Dicer substrates in correlation to both their binding affinity and cleavage efficiency. And by tertiary structural modeling constrained by icSHAPE-MaP RNA structural data, we find the spatial distance measuring as an influential parameter for Dicer cleavage-site selection.


Assuntos
RNA Helicases DEAD-box/metabolismo , Conformação de Ácido Nucleico , RNA/química , Ribonuclease III/metabolismo , Biologia Computacional , RNA Helicases DEAD-box/genética , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Mutagênese Sítio-Dirigida , Ligação Proteica/genética , RNA/genética , RNA/metabolismo , Sondas RNA , RNA-Seq , Ribonuclease III/genética , Especificidade por Substrato/genética
4.
Nat Commun ; 12(1): 3368, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099723

RESUMO

Folding of RNA can produce elaborate tertiary structures, corresponding to their diverse roles in the regulation of biological activities. Direct observation of RNA structures at high resolution in their native form however remains a challenge. The large vestibule and the narrow constriction of a Mycobacterium smegmatis porin A (MspA) suggests a sensing mode called nanopore trapping/translocation, which clearly distinguishes between microRNA, small interfering RNA (siRNA), transfer RNA (tRNA) and 5 S ribosomal RNA (rRNA). To further profit from the acquired event characteristics, a custom machine learning algorithm is developed. Events from measurements with a mixture of RNA analytes can be automatically classified, reporting a general accuracy of ~93.4%. tRNAs, which possess a unique tertiary structure, report a highly distinguishable sensing feature, different from all other RNA types tested in this study. With this strategy, tRNAs from different sources are measured and a high structural conservation across different species is observed in single molecule.


Assuntos
Mycobacterium smegmatis/metabolismo , Nanoporos , Porinas/metabolismo , RNA/metabolismo , Aprendizado de Máquina , MicroRNAs/química , MicroRNAs/genética , MicroRNAs/metabolismo , Simulação de Dinâmica Molecular , Peso Molecular , Mycobacterium smegmatis/genética , Conformação de Ácido Nucleico , Porinas/química , Porinas/genética , RNA/química , RNA/genética , Dobramento de RNA , Transporte de RNA , RNA Ribossômico 5S/química , RNA Ribossômico 5S/genética , RNA Ribossômico 5S/metabolismo , RNA Interferente Pequeno/química , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , RNA de Transferência/química , RNA de Transferência/genética , RNA de Transferência/metabolismo
5.
BMC Med Genomics ; 14(1): 155, 2021 06 11.
Artigo em Inglês | MEDLINE | ID: covidwho-1266486

RESUMO

BACKGROUND: COVID-19 is a respiratory viral infection with unique features including a more chronic course and systemic disease manifestations including multiple organ involvement; and there are differences in disease severity between ethnic groups. The immunological basis for disease has not been fully characterised. Analysis of whole-blood RNA expression may provide valuable information on disease pathogenesis. METHODS: We studied 45 patients with confirmed COVID-19 infection within 10 days from onset of illness and a control group of 19 asymptomatic healthy volunteers with no known exposure to COVID-19 in the previous 14 days. Relevant demographic and clinical information was collected and a blood sample was drawn from all participants for whole-blood RNA sequencing. We evaluated differentially-expressed genes in COVID-19 patients (log2 fold change ≥ 1 versus healthy controls; false-discovery rate < 0.05) and associated protein pathways and compared these to published whole-blood signatures for respiratory syncytial virus (RSV) and influenza. We developed a disease score reflecting the overall magnitude of expression of internally-validated genes and assessed the relationship between the disease score and clinical disease parameters. RESULTS: We found 135 differentially-expressed genes in the patients with COVID-19 (median age 35 years; 82% male; 36% Chinese, 53% South Asian ethnicity). Of the 117 induced genes, 14 were found in datasets from RSV and 40 from influenza; 95 genes were unique to COVID-19. Protein pathways were mostly generic responses to viral infections, including apoptosis by P53-associated pathway, but also included some unique pathways such as viral carcinogenesis. There were no major qualitative differences in pathways between ethnic groups. The composite gene-expression score was correlated with the time from onset of symptoms and nasal swab qPCR CT values (both p < 0.01) but was not related to participant age, gender, ethnicity or the presence or absence of chest X-ray abnormalities (all p > 0.05). CONCLUSIONS: The whole-blood transcriptome of COVID-19 has overall similarity with other respiratory infections but there are some unique pathways that merit further exploration to determine clinical relevance. The approach to a disease score may be of value, but needs further validation in a population with a greater range of disease severity.


Assuntos
COVID-19/patologia , RNA/sangue , Transcriptoma , Adulto , COVID-19/metabolismo , COVID-19/virologia , Portador Sadio/metabolismo , Portador Sadio/patologia , Feminino , Ontologia Genética , Humanos , Masculino , RNA/química , SARS-CoV-2/isolamento & purificação , Análise de Sequência de RNA , Regulação para Cima
6.
Nucleic Acids Res ; 49(11): 6587-6595, 2021 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-34107040

RESUMO

Bacteriophages have evolved a range of anti-CRISPR proteins (Acrs) to escape the adaptive immune system of prokaryotes, therefore Acrs can be used as switches to regulate gene editing. Herein, we report the crystal structure of a quaternary complex of AcrIIA14 bound SauCas9-sgRNA-dsDNA at 2.22 Å resolution, revealing the molecular basis for AcrIIA14 recognition and inhibition. Our structural and biochemical data analysis suggest that AcrIIA14 binds to a non-conserved region of SauCas9 HNH domain that is distinctly different from AcrIIC1 and AcrIIC3, with no significant effect on sgRNA or dsDNA binding. Further, our structural data shows that the allostery of the HNH domain close to the substrate DNA is sterically prevented by AcrIIA14 binding. In addition, the binding of AcrIIA14 triggers the conformational allostery of the HNH domain and the L1 linker within the SauCas9, driving them to make new interactions with the target-guide heteroduplex, enhancing the inhibitory ability of AcrIIA14. Our research both expands the current understanding of anti-CRISPRs and provides additional culues for the rational use of the CRISPR-Cas system in genome editing and gene regulation.


Assuntos
Proteínas de Bactérias/química , Proteína 9 Associada à CRISPR/antagonistas & inibidores , Proteína 9 Associada à CRISPR/química , Staphylococcus aureus/enzimologia , Regulação Alostérica , Cristalografia por Raios X , DNA/química , Modelos Moleculares , Domínios Proteicos , RNA/química
7.
Nucleic Acids Res ; 49(11): 6114-6127, 2021 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-34125895

RESUMO

Dynamic DNA nanodevices represent powerful tools for the interrogation and manipulation of biological systems. Yet, implementation remains challenging due to nuclease degradation and other cellular factors. Use of l-DNA, the nuclease resistant enantiomer of native d-DNA, provides a promising solution. On this basis, we recently developed a strand displacement methodology, referred to as 'heterochiral' strand displacement, that enables robust l-DNA nanodevices to be sequence-specifically interfaced with endogenous d-nucleic acids. However, the underlying reaction - strand displacement from PNA-DNA heteroduplexes - remains poorly characterized, limiting design capabilities. Herein, we characterize the kinetics of strand displacement from PNA-DNA heteroduplexes and show that reaction rates can be predictably tuned based on several common design parameters, including toehold length and mismatches. Moreover, we investigate the impact of nucleic acid stereochemistry on reaction kinetics and thermodynamics, revealing important insights into the biophysical mechanisms of heterochiral strand displacement. Importantly, we show that strand displacement from PNA-DNA heteroduplexes is compatible with RNA inputs, the most common nucleic acid target for intracellular applications. Overall, this work greatly improves the understanding of heterochiral strand displacement reactions and will be useful in the rational design and optimization of l-DNA nanodevices that operate at the interface with biology.


Assuntos
DNA/química , Ácidos Nucleicos Heteroduplexes/química , Ácidos Nucleicos Peptídicos/química , Cinética , RNA/química , Estereoisomerismo , Termodinâmica
8.
Methods Mol Biol ; 2277: 49-67, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34080144

RESUMO

Defects in human mitochondrial genome can cause a wide range of clinical disorders that still do not have efficient therapies. The natural pathway of small noncoding RNA import can be exploited to address therapeutic RNAs into the mitochondria. To create an approach of carrier-free targeting of RNA into living human cells, we designed conjugates containing a cholesterol residue and developed the protocols of chemical synthesis of oligoribonucleotides conjugated with cholesterol residue through cleavable pH-triggered hydrazone bond. The biodegradable conjugates of importable RNA with cholesterol can be internalized by cells in a carrier-free manner; RNA can then be released in the late endosomes due to a change in pH and partially targeted into mitochondria. Here we provide detailed protocols for solid-phase and "in solution" chemical synthesis of oligoribonucleotides conjugated to a cholesterol residue through a hydrazone bond. We describe the optimization of the carrier-free cell transfection with these conjugated RNA molecules and methods for evaluating the cellular and mitochondrial uptake of lipophilic conjugates.


Assuntos
Mitocôndrias/genética , Oligorribonucleotídeos/síntese química , RNA/química , Transfecção/métodos , Células Cultivadas , Colesterol/química , Humanos , Hidrazonas/química , Concentração de Íons de Hidrogênio , RNA/administração & dosagem
9.
BMC Med Genomics ; 14(1): 155, 2021 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-34116667

RESUMO

BACKGROUND: COVID-19 is a respiratory viral infection with unique features including a more chronic course and systemic disease manifestations including multiple organ involvement; and there are differences in disease severity between ethnic groups. The immunological basis for disease has not been fully characterised. Analysis of whole-blood RNA expression may provide valuable information on disease pathogenesis. METHODS: We studied 45 patients with confirmed COVID-19 infection within 10 days from onset of illness and a control group of 19 asymptomatic healthy volunteers with no known exposure to COVID-19 in the previous 14 days. Relevant demographic and clinical information was collected and a blood sample was drawn from all participants for whole-blood RNA sequencing. We evaluated differentially-expressed genes in COVID-19 patients (log2 fold change ≥ 1 versus healthy controls; false-discovery rate < 0.05) and associated protein pathways and compared these to published whole-blood signatures for respiratory syncytial virus (RSV) and influenza. We developed a disease score reflecting the overall magnitude of expression of internally-validated genes and assessed the relationship between the disease score and clinical disease parameters. RESULTS: We found 135 differentially-expressed genes in the patients with COVID-19 (median age 35 years; 82% male; 36% Chinese, 53% South Asian ethnicity). Of the 117 induced genes, 14 were found in datasets from RSV and 40 from influenza; 95 genes were unique to COVID-19. Protein pathways were mostly generic responses to viral infections, including apoptosis by P53-associated pathway, but also included some unique pathways such as viral carcinogenesis. There were no major qualitative differences in pathways between ethnic groups. The composite gene-expression score was correlated with the time from onset of symptoms and nasal swab qPCR CT values (both p < 0.01) but was not related to participant age, gender, ethnicity or the presence or absence of chest X-ray abnormalities (all p > 0.05). CONCLUSIONS: The whole-blood transcriptome of COVID-19 has overall similarity with other respiratory infections but there are some unique pathways that merit further exploration to determine clinical relevance. The approach to a disease score may be of value, but needs further validation in a population with a greater range of disease severity.


Assuntos
COVID-19/patologia , RNA/sangue , Transcriptoma , Adulto , COVID-19/metabolismo , COVID-19/virologia , Portador Sadio/metabolismo , Portador Sadio/patologia , Feminino , Ontologia Genética , Humanos , Masculino , RNA/química , SARS-CoV-2/isolamento & purificação , Análise de Sequência de RNA , Regulação para Cima
10.
Nucleic Acids Res ; 49(W1): W685-W690, 2021 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-34107038

RESUMO

We present an updated version of the Voronoia service that enables fully automated analysis of the atomic packing density of macromolecules. Voronoia combines previous efforts to analyse 3D protein and RNA structures into a single service, combined with state-of-the-art online visualization. Voronoia uses the Voronoi cell method to calculate the free space between neighbouring atoms to estimate van der Waals interactions. Compared to other methods that derive van der Waals interactions by calculating solvent-free surfaces, it explicitly considers volume or packing defects. Large internal voids refer either to water molecules or ions unresolved by X-ray crystallography or cryo-EM, cryptic ligand binding pockets, or parts of a structural model that require further refinement. Voronoia is, therefore mainly used for functional analyses of 3D structures and quality assessments of structural models. Voronoia 4-ever updates the database of precomputed packing densities of PDB entries, allows uploading multiple structures, adds new filter options and facilitates direct access to the results through intuitive display with the NGL viewer. Voronoia is available at: htttp://proteinformatics.org/voronoia.


Assuntos
Conformação Proteica , Software , Modelos Moleculares , RNA/química
11.
Nat Commun ; 12(1): 3877, 2021 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-34162884

RESUMO

Methylation is a prevalent post-transcriptional modification encountered in coding and non-coding RNA. For RNA methylation, cells use methyltransferases and small organic substances as methyl-group donors, such as S-adenosylmethionine (SAM). SAM and other nucleotide-derived cofactors are viewed as evolutionary leftovers from an RNA world, in which riboswitches have regulated, and ribozymes have catalyzed essential metabolic reactions. Here, we disclose the thus far unrecognized direct link between a present-day riboswitch and its inherent reactivity for site-specific methylation. The key is O6-methyl pre-queuosine (m6preQ1), a potentially prebiotic nucleobase which is recognized by the native aptamer of a preQ1 class I riboswitch. Upon binding, the transfer of the ligand's methyl group to a specific cytidine occurs, installing 3-methylcytidine (m3C) in the RNA pocket under release of pre-queuosine (preQ1). Our finding suggests that nucleic acid-mediated methylation is an ancient mechanism that has offered an early path for RNA epigenetics prior to the evolution of protein methyltransferases. Furthermore, our findings may pave the way for the development of riboswitch-descending methylation tools based on rational design as a powerful alternative to in vitro selection approaches.


Assuntos
Conformação de Ácido Nucleico , Nucleosídeo Q/química , RNA/química , Riboswitch , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/genética , Aptâmeros de Nucleotídeos/metabolismo , Sequência de Bases , Cinética , Metilação , Estrutura Molecular , Nucleosídeo Q/metabolismo , RNA/genética , RNA/metabolismo , S-Adenosilmetionina/química , S-Adenosilmetionina/metabolismo
12.
Nucleic Acids Res ; 49(W1): W505-W509, 2021 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-34060619

RESUMO

Prime editing (PE) is a novel CRISPR-derived genome editing technique facilitating precision editing without double-stranded DNA breaks. PE, mediated by a Cas9-reverse transcriptase fusion protein, is based on dual-functioning prime editing guide RNAs (pegRNAs), serving both as guide molecules and as templates carrying the desired edits. Due to such diverse functions, manual pegRNA design is a subject to error and not suited for large-scale setups. Here, we present pegIT, a user-friendly web tool for rapid pegRNA design for numerous user-defined edits, including large-scale setups. pegIT is freely available at https://pegit.giehmlab.dk.


Assuntos
Edição de Genes/métodos , Software , Proteína 9 Associada à CRISPR/genética , Internet , RNA/química , DNA Polimerase Dirigida por RNA/genética , Proteínas Recombinantes de Fusão
13.
Int J Mol Sci ; 22(9)2021 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-34066402

RESUMO

Newly designed and synthesized diarylethene (DAE) derivatives with aliphatic amine sidearms and one with two pyrenes, revealed excellent photo-switching property of central DAE core in MeOH and water. The only exception was bis-pyrene analogue, its DAE core very readily photochemically closed, but reversible opening completely hampered by aromatic stacking interaction of pyrene(s) with cyclic DAE. In this process, pyrene fluorescence showed to be a reliable monitoring method, an open form characterized by strong emission at 480 nm (typical for pyrene-aggregate), while closed form emitted weakly at 400 nm (typical for pyrene-DAE quenching). Only open DAE-bis-pyrene form interacted measurably with ds-DNA/RNA by flexible insertion in polynucleotide grooves, while self-stacked closed form did not bind to DNA/RNA. For the same steric reasons, flexible open DAE-bis-pyrene form was bound to at least three different binding sites at bovine serum albumin (BSA), while rigid, self-stacked closed form interacted dominantly with only one BSA site. Preliminary screening of antiproliferative activity against human lung carcinoma cell line A549 revealed that all DAE-derivatives are non-toxic. However, bis-pyrene analogue efficiently entered cells and located in the cytoplasm, whereby irradiation by light (315-400 nm) resulted in a strong, photo-induced cytotoxic effect, typical for pyrene-related singlet oxygen species production.


Assuntos
DNA/química , Luz , Conformação Molecular , Pirenos/química , Soroalbumina Bovina/química , Células A549 , Animais , Bovinos , Sobrevivência Celular , Elétrons , Humanos , Desnaturação de Ácido Nucleico/efeitos da radiação , Processos Fotoquímicos , RNA/química , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta , Temperatura
14.
Molecules ; 26(11)2021 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-34070959

RESUMO

Although natural or artificial modified pyrimidine nucleobases represent important molecules with valuable properties as constituents of DNA and RNA, no systematic analyses of the structural aspects of bromo derivatives of cytosine have appeared so far in the literature. In view of the biochemical and pharmaceutical relevance of these compounds, six different crystals containing proton-transfer derivatives of 5-bromocytosine are prepared and analyzed in the solid-state by single crystal X-ray diffraction. All six compounds are organic salts, with proton transfer occurring to the Nimino atom of the pyridine ring. Experimental results are then complemented with Hirshfeld surface analysis to quantitively evaluate the contribution of different intermolecular interactions in the crystal packing. Furthermore, theoretical calculations, based on different arrangements of molecules extracted from the crystal structure determinations, are carried out to analyze the formation mechanism of halogen bonds (XBs) in these compounds and provide insights into the nature and strength of the observed interactions. The results show that the supramolecular architectures of the six molecular salts involve extensive classical intermolecular hydrogen bonds. However, in all but one proton-transfer adducts, weak to moderate XBs are revealed by C-Br…O short contacts between the bromine atom in the fifth position, which acts as XB donor (electron acceptor). Moreover, the lone pair electrons of the oxygen atom of adjacent pyrimidine nucleobases and/or counterions or water molecules, which acts as XB acceptor (electron donor).


Assuntos
Citosina/análogos & derivados , Cristalografia por Raios X/métodos , Cisteína/química , Citosina/química , Citosina/metabolismo , DNA/química , Elétrons , Halogênios/química , Hidrogênio/química , Ligação de Hidrogênio , Modelos Moleculares , Prótons , Piridinas/química , RNA/química , Difração de Raios X/métodos
15.
Nat Commun ; 12(1): 3549, 2021 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-34112799

RESUMO

Fluorogenic RNA aptamers are synthetic functional RNAs that specifically bind and activate conditional fluorophores. The Chili RNA aptamer mimics large Stokes shift fluorescent proteins and exhibits high affinity for 3,5-dimethoxy-4-hydroxybenzylidene imidazolone (DMHBI) derivatives to elicit green or red fluorescence emission. Here, we elucidate the structural and mechanistic basis of fluorescence activation by crystallography and time-resolved optical spectroscopy. Two co-crystal structures of the Chili RNA with positively charged DMHBO+ and DMHBI+ ligands revealed a G-quadruplex and a trans-sugar-sugar edge G:G base pair that immobilize the ligand by π-π stacking. A Watson-Crick G:C base pair in the fluorophore binding site establishes a short hydrogen bond between the N7 of guanine and the phenolic OH of the ligand. Ultrafast excited state proton transfer (ESPT) from the neutral chromophore to the RNA was found with a time constant of 130 fs and revealed the mode of action of the large Stokes shift fluorogenic RNA aptamer.


Assuntos
Aptâmeros de Nucleotídeos/química , Quadruplex G , Guanina/química , RNA/química , Sítios de Ligação , Cristalografia , Fluorescência , Ligação de Hidrogênio , Ligantes , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Mutagênese , Mutação , Prótons , Espectrometria de Fluorescência
16.
Nat Commun ; 12(1): 3494, 2021 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-34108470

RESUMO

Non-coding RNAs (ncRNA) are essential for all life, and their functions often depend on their secondary (2D) and tertiary structure. Despite the abundance of software for the visualisation of ncRNAs, few automatically generate consistent and recognisable 2D layouts, which makes it challenging for users to construct, compare and analyse structures. Here, we present R2DT, a method for predicting and visualising a wide range of RNA structures in standardised layouts. R2DT is based on a library of 3,647 templates representing the majority of known structured RNAs. R2DT has been applied to ncRNA sequences from the RNAcentral database and produced >13 million diagrams, creating the world's largest RNA 2D structure dataset. The software is amenable to community expansion, and is freely available at https://github.com/rnacentral/R2DT and a web server is found at https://rnacentral.org/r2dt .


Assuntos
Biologia Computacional/métodos , RNA/química , Bases de Dados de Ácidos Nucleicos , Conformação de Ácido Nucleico , RNA não Traduzido/química , Reprodutibilidade dos Testes , Análise de Sequência de RNA , Software
17.
Nucleic Acids Res ; 49(W1): W499-W504, 2021 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-33939828

RESUMO

Prime editing technology is capable of generating targeted insertions, deletions, and base conversions. However, the process of designing prime editing guide RNAs (pegRNAs), which contain a primer binding site and a reverse-transcription template at the 3' end, is more complex than that for the single guide RNAs used with CRISPR nucleases or base editors. Furthermore, the assessment of high-throughput sequencing data after prime editors (PEs) have been employed should consider the unique feature of PEs; thus, pre-existing assessment tools cannot directly be adopted for PEs. Here, we present two user-friendly web-based tools for PEs, named PE-Designer and PE-Analyzer. PE-Designer, a dedicated tool for pegRNA selection, provides all possible target sequences, pegRNA extension sequences, and nicking guide RNA sequences together with useful information, and displays the results in an interactive image. PE-Analyzer, a dedicated tool for PE outcome analysis, accepts high-throughput sequencing data, summarizes mutation-related information in a table, and provides interactive graphs. PE-Analyzer was mainly written using JavaScript so that it can analyze several data sets without requiring that huge sequencing data (>100MB) be uploaded to the server, reducing analysis time and increasing personal security. PE-Designer and PE-Analyzer are freely available at http://www.rgenome.net/pe-designer/ and http://www.rgenome.net/pe-analyzer/ without a login process.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Software , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Internet , Mutação , RNA/química , Alinhamento de Sequência
18.
Nucleic Acids Res ; 49(W1): W491-W498, 2021 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-34009383

RESUMO

OxDNA and oxRNA are popular coarse-grained models used by the DNA/RNA nanotechnology community to prototype, analyze and rationalize designed DNA and RNA nanostructures. Here, we present oxDNA.org, a graphical web interface for running, visualizing and analyzing oxDNA and oxRNA molecular dynamics simulations on a GPU-enabled high performance computing server. OxDNA.org automatically generates simulation files, including a multi-step relaxation protocol for structures exported in non-physical states from DNA/RNA design tools. Once the simulation is complete, oxDNA.org provides an interactive visualization and analysis interface using the browser-based visualizer oxView to facilitate the understanding of simulation results for a user's specific structure. This online tool significantly lowers the entry barrier of integrating simulations in the nanostructure design pipeline for users who are not experts in the technical aspects of molecular simulation. The webserver is freely available at oxdna.org.


Assuntos
DNA/química , Simulação de Dinâmica Molecular , Nanoestruturas/química , RNA/química , Software , Internet , Conformação de Ácido Nucleico
19.
Nucleic Acids Res ; 49(W1): W530-W534, 2021 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-33950214

RESUMO

With the growth of protein structure data, the analysis of molecular interactions between ligands and their target molecules is gaining importance. PLIP, the protein-ligand interaction profiler, detects and visualises these interactions and provides data in formats suitable for further processing. PLIP has proven very successful in applications ranging from the characterisation of docking experiments to the assessment of novel ligand-protein complexes. Besides ligand-protein interactions, interactions with DNA and RNA play a vital role in many applications, such as drugs targeting DNA or RNA-binding proteins. To date, over 7% of all 3D structures in the Protein Data Bank include DNA or RNA. Therefore, we extended PLIP to encompass these important molecules. We demonstrate the power of this extension with examples of a cancer drug binding to a DNA target, and an RNA-protein complex central to a neurological disease. PLIP is available online at https://plip-tool.biotec.tu-dresden.de and as open source code. So far, the engine has served over a million queries and the source code has been downloaded several thousand times.


Assuntos
DNA/química , Proteínas de Ligação a RNA/química , RNA/química , Software , Algoritmos , Antineoplásicos/química , Guanosina Trifosfato/química , Ligantes , Conformação de Ácido Nucleico , Fenazinas/química , Conformação Proteica , RNA Polimerase II/química , Elementos de Resposta
20.
Nat Commun ; 12(1): 3018, 2021 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-34021158

RESUMO

High-yielding and selective prebiotic syntheses of RNA and DNA nucleotides involve UV irradiation to promote the key reaction steps and eradicate biologically irrelevant isomers. While these syntheses were likely enabled by UV-rich prebiotic environment, UV-induced formation of photodamages in polymeric nucleic acids, such as cyclobutane pyrimidine dimers (CPDs), remains the key unresolved issue for the origins of RNA and DNA on Earth. Here, we demonstrate that substitution of adenine with 2,6-diaminopurine enables repair of CPDs with yields reaching 92%. This substantial self-repairing activity originates from excellent electron donating properties of 2,6-diaminopurine in nucleic acid strands. We also show that the deoxyribonucleosides of 2,6-diaminopurine and adenine can be formed under the same prebiotic conditions. Considering that 2,6-diaminopurine was previously shown to increase the rate of nonenzymatic RNA replication, this nucleobase could have played critical roles in the formation of functional and photostable RNA/DNA oligomers in UV-rich prebiotic environments.


Assuntos
2-Aminopurina/análogos & derivados , 2-Aminopurina/farmacologia , Reparo do DNA/efeitos dos fármacos , DNA/efeitos dos fármacos , Prebióticos , Adenina , DNA/efeitos da radiação , Simulação de Dinâmica Molecular , Ácidos Nucleicos , Nucleotídeos , Dímeros de Pirimidina , RNA/química , Raios Ultravioleta/efeitos adversos
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