RESUMO
Visualizing the physical basis for molecular behaviour inside living cells is a great challenge for biology. RNAs are central to biological regulation, and the ability of RNA to adopt specific structures intimately controls every step of the gene expression program. However, our understanding of physiological RNA structures is limited; current in vivo RNA structure profiles include only two of the four nucleotides that make up RNA. Here we present a novel biochemical approach, in vivo click selective 2'-hydroxyl acylation and profiling experiment (icSHAPE), which enables the first global view, to our knowledge, of RNA secondary structures in living cells for all four bases. icSHAPE of the mouse embryonic stem cell transcriptome versus purified RNA folded in vitro shows that the structural dynamics of RNA in the cellular environment distinguish different classes of RNAs and regulatory elements. Structural signatures at translational start sites and ribosome pause sites are conserved from in vitro conditions, suggesting that these RNA elements are programmed by sequence. In contrast, focal structural rearrangements in vivo reveal precise interfaces of RNA with RNA-binding proteins or RNA-modification sites that are consistent with atomic-resolution structural data. Such dynamic structural footprints enable accurate prediction of RNA-protein interactions and N(6)-methyladenosine (m(6)A) modification genome wide. These results open the door for structural genomics of RNA in living cells and reveal key physiological structures controlling gene expression.
Assuntos
Regulação da Expressão Gênica , Conformação de Ácido Nucleico , RNA/química , RNA/genética , Acilação , Adenosina/análogos & derivados , Animais , Sítios de Ligação , Sobrevivência Celular , Química Click , Biologia Computacional , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica/genética , Genoma/genética , Camundongos , Modelos Moleculares , Biossíntese de Proteínas/genética , RNA/classificação , RNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Sequências Reguladoras de Ácido Ribonucleico/genética , Ribossomos/metabolismo , Transcriptoma/genéticaRESUMO
Capillary electrophoresis (CE) is a powerful analytical tool for performing separations and characterizing properties of charged species. For reacting species during a CE separation, local concentrations change leading to nonequilibrium conditions. Interpreting experimental data with such nonequilibrium reactive species is nontrivial due to the large number of variables involved in the system. In this work we develop a COMSOL multiphysics-based numerical model to simulate the electrokinetic mass transport of short interacting ssDNAs in microchip capillary electrophoresis. We probe the importance of the dissociation constant, K(D), and the concentration of DNA on the resulting observed mobility of the dsDNA peak, µ(w), by using a full sweep of parametric simulations. We find that the observed mobility is strongly dependent on the DNA concentration and K(D), as well as ssDNA concentration, and develop a relation with which to understand this dependence. Furthermore, we present experimental microchip capillary electrophoresis measurements of interacting 10 base ssDNA and its complement with changes in buffer ionic strength, DNA concentration, and DNA sequence to vary the system equilibria. We then compare our results to thermodynamically calculated K(D) values.
Assuntos
DNA/análise , DNA/química , Eletroforese Capilar/métodos , Eletroforese em Microchip/métodos , Hibridização de Ácido Nucleico/métodos , Modelos Teóricos , Concentração Osmolar , TermodinâmicaRESUMO
RNA structure has important roles in practically every facet of gene regulation, but the paucity of in vivo structural probes limits current understanding. Here we design, synthesize and demonstrate two new chemical probes that enable selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) in living cells. RNA structures in human, mouse, fly, yeast and bacterial cells are read out at single-nucleotide resolution, revealing tertiary contacts and RNA-protein interactions.
Assuntos
Conformação de Ácido Nucleico , RNA/química , Acilação , Animais , Humanos , Modelos MolecularesRESUMO
Heavy metal contamination of water can be toxic to humans and wildlife; thus the development of methods to detect this contamination is of high importance. Here we describe the design and application of DNA-based fluorescent chemosensors on microbeads to differentiate eight toxic metal ions in water. We developed and synthesized four fluorescent 2'-deoxyribosides of metal-binding ligands. A tetramer-length oligodeoxy-fluoroside (ODF) library of 6561 members was constructed and screened for sequences responsive to metal ions, of which seven sequences were selected. Statistical analysis of the response patterns showed successful differentiation of the analytes at concentrations as low as 100â nM. Sensors were able to classify water samples from 13 varied sites and quantify metal contamination in unknown specimens. The results demonstrate the practical potential of bead-based ODF chemosensors to analyze heavy metal contamination in water samples by a simple and inexpensive optical method.
Assuntos
Água Doce/química , Metais Pesados/análise , Oligonucleotídeos/química , Análise Discriminante , Monitoramento Ambiental , Quinolinas/química , Espectrometria de FluorescênciaRESUMO
Kinetics studies with structurally varied aldehydes and ketones in aqueous buffer at pH 7.4 reveal that carbonyl compounds with neighboring acid/base groups form hydrazones at accelerated rates. Similarly, tests of a hydrazine with a neighboring carboxylic acid group show that it also reacts at an accelerated rate. Rate constants for the fastest carbonyl/hydrazine combinations are 2-20 M(-1) s(-1), which is faster than recent strain-promoted cycloaddition reactions.
Assuntos
Hidrazonas/química , Aldeídos/química , Soluções Tampão , Concentração de Íons de Hidrogênio , Cetonas/química , CinéticaRESUMO
The formation of oximes and hydrazones is widely used in chemistry and biology as a molecular conjugation strategy for achieving ligation, attachment, and bioconjugation. However, the relatively slow rate of reaction has hindered its utility. Here, we report that simple, commercially available anthranilic acids and aminobenzoic acids act as superior catalysts for hydrazone and oxime formation, speeding the reaction considerably over the traditional aniline-catalyzed reaction at neutral pH. This efficient nucleophilic catalysis, involving catalyst-imine intermediates, allows rapid hydrazone/oxime formation even with relatively low concentrations of the two reactants. The most efficient catalysts are found to be 5-methoxyanthranilic acid and 3,5-diaminobenzoic acid; we find that they can enhance rates by factors of as much as 1-2 orders of magnitude over the aniline-catalyzed reaction. Evidence based on a range of differently substituted arylamines suggests that the ortho-carboxylate group in the anthranilate catalysts serves to aid in intramolecular proton transfer during imine and hydrazone formation.
Assuntos
Aminobenzoatos/química , Hidrazonas/química , Oximas/química , Água/química , meta-Aminobenzoatos/química , Catálise , Concentração de Íons de Hidrogênio , Estrutura Molecular , PrótonsRESUMO
We describe the synthesis and properties of new fluorescence quenchers containing aldehyde, hydrazine, and aminooxy groups, allowing convenient bioconjugation as oximes or hydrazones. Conjugation to oligonucleotides proceeded in high yield with aniline as catalyst. Kinetics studies of conjugation show that, under optimal conditions, a hydrazine or aminooxy quencher can react with aldehyde-modified DNA to form a stable hydrazone or oxime adduct in as little as five minutes. The resulting quencher-containing DNAs were assessed for their ability to quench the emission of fluorescein in labeled complements and compared to the commercially available dabcyl and Black Hole Quencher 2 (BHQ2), which were conjugated as phosphoramidites. Results show that the new quenchers possess slightly different absorbance properties compared to dabcyl and are as efficient as the commercial quenchers in quenching fluorescein emission. Hydrazone-based quenchers were further successfully incorporated into molecular beacons and shown to give high signal to background ratios in single nucleotide polymorphism detection in vitro. Finally, aminooxy and hydrazine quenchers were applied to quenching of an aldehyde-containing fluorophore associated with living cells, demonstrating cellular quenching within one hour.
Assuntos
Fluorescência , Hidrazonas/química , Oximas/química , DNA/química , Cinética , Espectroscopia de Ressonância Magnética , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Fluorescence quenching groups are widely employed in biological detection, sensing, and imaging. To date, a relatively small number of such groups are in common use. Perhaps the most commonly used quencher, dabcyl, has limited efficiency with a broad range of fluorophores. Here, we describe a molecular approach to improve the efficiency of quenchers by increasing their electronic complexity. Multi-Path Quenchers (MPQ) are designed to have multiple donor or acceptor groups in their structure, allowing for a multiplicity of conjugation pathways of varied length. This has the effect of broadening the absorption spectrum, which in turn can increase quenching efficiency and versatility. Six such MPQ derivatives are synthesized and tested for quenching efficiency in a DNA hybridization context. Duplexes placing quenchers and fluorophores within contact distance or beyond this distance are used to measure quenching via contact or FRET mechanisms. Results show that several of the quenchers are considerably more efficient than dabcyl at quenching a wider range of common fluorophores, and two quench fluorescein and TAMRA as well as or better than a Black Hole Quencher.
Assuntos
Corantes Fluorescentes/química , Espectrometria de Fluorescência/métodos , Sequência de Bases , DNA/química , Transferência Ressonante de Energia de Fluorescência/métodos , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular/métodosRESUMO
We develop surface-modified 100 nm silica nanofluidic channels that change in measured conductivity upon exposure to single- or double-stranded DNA. Through careful monitoring of both electromigrative and advective current in the channel, we can detect nanomolar concentrations of DNA. These results can be exploited for inexpensive, all-electronic DNA sensors.
Assuntos
DNA de Cadeia Simples/química , DNA/química , Nanoestruturas/química , Dióxido de Silício/química , Propriedades de SuperfícieRESUMO
Hydrazones and oximes are widely useful structures for conjugate formation in chemistry and biology, but their formation can be slow at neutral pH. Kinetics studies were performed for a range of structurally varied hydrazines, and a surprisingly large variation in reaction rate was observed. Structures that undergo especially rapid reactions were identified, enabling reaction rates that rival orthogonal cycloaddition-based conjugation chemistries.
Assuntos
Hidrazonas/química , Oximas/química , Catálise , Técnicas de Química Combinatória , Hidrazinas/química , Concentração de Íons de Hidrogênio , Cinética , Estrutura MolecularRESUMO
Anthranilic acids were recently reported as superior catalysts for hydrazone and oxime formation compared to aniline, the classic catalyst for these reactions. Here, alternative proton donors were examined with varied pKa in an effort to enhance activity at biological pH. The experiments show that 2-aminobenzenephosphonic acids are superior to anthranilic acids in catalyzing hydrazone formation with common aldehyde substrates.