RESUMO
Transcription in RNA viruses is highly dynamic, with a variety of pauses interrupting nucleotide addition by RNA-dependent RNA polymerase (RdRp). For example, rare but lengthy pauses (>20 s) have been linked to backtracking for viral single-subunit RdRps. However, while such backtracking has been well characterized for multi-subunit RNA polymerases (RNAPs) from bacteria and yeast, little is known about the details of viral RdRp backtracking and its biological roles. Using high-throughput magnetic tweezers, we quantify the backtracking by RdRp from the double-stranded (ds) RNA bacteriophage Φ6, a model system for RdRps. We characterize the probability of entering long backtracks as a function of force and propose a model in which the bias toward backtracking is determined by the base paring at the dsRNA fork. We further discover that extensive backtracking provides access to a new 3'-end that allows for the de novo initiation of a second RdRp. This previously unidentified behavior provides a new mechanism for rapid RNA synthesis using coupled RdRps and hints at a possible regulatory pathway for gene expression during viral RNA transcription.
Assuntos
Bacteriófago phi 6/enzimologia , RNA Polimerase Dependente de RNA/metabolismo , Sítio de Iniciação de Transcrição , Moldes Genéticos , Transcrição GênicaRESUMO
BACKGROUND: Zero-mode waveguides (ZMWs) are photonic nanostructures that create highly confined optical observation volumes, thereby allowing single-molecule-resolved biophysical studies at relatively high concentrations of fluorescent molecules. This principle has been successfully applied in single-molecule, real-time (SMRT®) DNA sequencing for the detection of DNA sequences and DNA base modifications. In contrast, RNA sequencing methods cannot provide sequence and RNA base modifications concurrently as they rely on complementary DNA (cDNA) synthesis by reverse transcription followed by sequencing of cDNA. Thus, information on RNA modifications is lost during the process of cDNA synthesis. RESULTS: Here we describe an application of SMRT technology to follow the activity of reverse transcriptase enzymes synthesizing cDNA on thousands of single RNA templates simultaneously in real time with single nucleotide turnover resolution using arrays of ZMWs. This method thereby obtains information from the RNA template directly. The analysis of the kinetics of the reverse transcriptase can be used to identify RNA base modifications, shown by example for N6-methyladenine (m6A) in oligonucleotides and in a specific mRNA extracted from total cellular mRNA. Furthermore, the real-time reverse transcriptase dynamics informs about RNA secondary structure and its rearrangements, as demonstrated on a ribosomal RNA and an mRNA template. CONCLUSIONS: Our results highlight the feasibility of studying RNA modifications and RNA structural rearrangements in ZMWs in real time. In addition, they suggest that technology can be developed for direct RNA sequencing provided that the reverse transcriptase is optimized to resolve homonucleotide stretches in RNA.
Assuntos
Nanotecnologia/métodos , RNA Mensageiro/análise , Transcrição Reversa , DNA Complementar/análise , DNA Complementar/genética , Rearranjo Gênico , Cinética , Nanoestruturas/química , Nucleotídeos/química , RNA Mensageiro/química , RNA Mensageiro/genética , DNA Polimerase Dirigida por RNA/genética , DNA Polimerase Dirigida por RNA/metabolismo , Análise de Sequência de DNA/métodosRESUMO
Solid-state nanopores can be employed to detect and study local structure along single molecules by voltage driven translocation through the nanopore. Their sensitivity and versatility can be augmented by combining them with a direct force probe, for example, optical tweezers. Such a tool could potentially be used to directly probe RNA secondary structure through the sequential unfolding of duplex regions. Here, we demonstrate the first application of such a system to the study of RNA by directly measuring the net force on individual double-stranded RNA (dsRNA) molecules. We have probed the force on dsRNA over a large range of nanopore sizes from 35 nm down to 3.5 nm and find that it decreases as the pore size is increased, in accordance with numerical calculations. Furthermore, we find that the force is independent of the distance between the optical trap and the nanopore surface, permitting force measurement on quite short molecules. By comparison with dsDNA molecules trapped in the same nanopores, we find that the force on dsRNA is on the order of, but slightly lower than, that on dsDNA. With these measurements, we expand the possibilities of the nanopore-optical tweezers to the study of RNA molecules with potential applications to the detection of RNA-bound proteins, the determination of RNA secondary structure, and the processing of RNA by molecular motors.
Assuntos
Biofísica/métodos , Nanoestruturas/química , Nanotecnologia/métodos , RNA de Cadeia Dupla/química , RNA Viral/química , Biofísica/instrumentação , Biotina/química , Primers do DNA/química , Desenho de Equipamento , Teste de Materiais , Nanopartículas/química , Pinças Ópticas , Reação em Cadeia da Polimerase/métodos , Poliestirenos/química , Estreptavidina/químicaRESUMO
RNA-dependent RNA polymerases (RdRP) form an important class of enzymes that is responsible for genome replication and transcription in RNA viruses and involved in the regulation of RNA interference in plants and fungi. The RdRP kinetics have been extensively studied, but pausing, an important regulatory mechanism for RNA polymerases that has also been implicated in RNA recombination, has not been considered. Here, we report that RdRP experience a dramatic, long-lived decrease in its elongation rate when it is reinitiated following stalling. The rate decrease has an intriguingly weak temperature dependence, is independent of both the nucleotide concentration during stalling and the length of the RNA transcribed prior to stalling; however it is sensitive to RNA structure. This allows us to delineate the potential factors underlying this irreversible conversion of the elongation complex to a less active mode.
Assuntos
Bacteriófago phi 6/enzimologia , RNA Polimerase Dependente de RNA/metabolismo , RNA/biossíntese , Proteínas Virais/metabolismo , Bacteriófago phi 6/fisiologia , Cinética , Nucleotídeos/metabolismo , RNA/química , Temperatura , Transcrição Gênica , Replicação ViralRESUMO
Precise, controllable single-molecule force spectroscopy studies of RNA and RNA-dependent processes have recently shed new light on the dynamics and pathways of RNA folding and RNA-enzyme interactions. A crucial component of this research is the design and assembly of an appropriate RNA construct. Such a construct is typically subject to several criteria. First, single-molecule force spectroscopy techniques often require an RNA construct that is longer than the RNA molecules used for bulk biochemical studies. Next, the incorporation of modified nucleotides into the RNA construct is required for its surface immobilization. In addition, RNA constructs for single-molecule studies are commonly assembled from different single-stranded RNA molecules, demanding good control of hybridization or ligation. Finally, precautions to prevent RNase- and divalent cation-dependent RNA digestion must be taken. The rather limited selection of molecular biology tools adapted to the manipulation of RNA molecules, as well as the sensitivity of RNA to degradation, make RNA construct preparation a challenging task. We briefly illustrate the types of single-molecule force spectroscopy experiments that can be performed on RNA, and then present an overview of the toolkit of molecular biology techniques at one's disposal for the assembly of such RNA constructs. Within this context, we evaluate the molecular biology protocols in terms of their effectiveness in producing long and stable RNA constructs.
Assuntos
RNA/química , Análise Espectral/métodos , Pinças Ópticas , RNA/biossíntese , RNA/isolamento & purificação , RNA de Cadeia Dupla/biossíntese , RNA de Cadeia Dupla/química , Transcrição GênicaRESUMO
Thousands of pathogens are known to infect humans, but only a fraction are readily identifiable using current diagnostic methods. Microbial cell-free DNA sequencing offers the potential to non-invasively identify a wide range of infections throughout the body, but the challenges of clinical-grade metagenomic testing must be addressed. Here we describe the analytical and clinical validation of a next-generation sequencing test that identifies and quantifies microbial cell-free DNA in plasma from 1,250 clinically relevant bacteria, DNA viruses, fungi and eukaryotic parasites. Test accuracy, precision, bias and robustness to a number of metagenomics-specific challenges were determined using a panel of 13 microorganisms that model key determinants of performance in 358 contrived plasma samples, as well as 2,625 infections simulated in silico and 580 clinical study samples. The test showed 93.7% agreement with blood culture in a cohort of 350 patients with a sepsis alert and identified an independently adjudicated cause of the sepsis alert more often than all of the microbiological testing combined (169 aetiological determinations versus 132). Among the 166 samples adjudicated to have no sepsis aetiology identified by any of the tested methods, sequencing identified microbial cell-free DNA in 62, likely derived from commensal organisms and incidental findings unrelated to the sepsis alert. Analysis of the first 2,000 patient samples tested in the CLIA laboratory showed that more than 85% of results were delivered the day after sample receipt, with 53.7% of reports identifying one or more microorganisms.
Assuntos
Ácidos Nucleicos Livres/genética , Doenças Transmissíveis/diagnóstico , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Estudos de Coortes , Doenças Transmissíveis/microbiologia , Doenças Transmissíveis/parasitologia , Doenças Transmissíveis/virologia , DNA Bacteriano/genética , DNA Fúngico/genética , DNA Viral/genética , Humanos , Sepse/diagnóstico , Sepse/microbiologiaRESUMO
The binding of ciprofloxacin to natural and synthetic polymeric DNAs was investigated at different solvent conditions using a combination of spectroscopic and hydrodynamic techniques. In 10 mM cacodylate buffer (pH 7.0) containing 108.6 mM Na(+), no sequence preferences in the interaction of ciprofloxacin with DNA was detected, while in 2 mM cacodylate buffer (pH 7.0) containing only 1.7 mM Na(+), a significant binding of ciprofloxacin to natural and synthetic linear double-stranded DNA was observed. At low ionic strength of solution, ciprofloxacin binding to DNA duplex containing alternating AT base pairs is accompanied by the largest enhancement in thermal stability (e.g. DeltaT(m) approximately 10 degrees C for poly[d(AT)].poly[d(AT)]), and the most pronounced red shift in the position of the maximum of the fluorescence emission spectrum (lambda(max)). Similar red shift in the position of lambda(max) is also observed for ciprofloxacin binding to dodecameric duplex containing five successive alternating AT base pairs in the row. On the other hand, ciprofloxacin binding to poly[d(GC)].poly[d(GC)], calf thymus DNA and dodecameric duplex containing a mixed sequence is accompanied by the largest fluorescence intensity quenching. Addition of NaCl does not completely displace ciprofloxacin bound to DNA, indicating the binding is not entirely electrostatic in origin. The intrinsic viscosity data suggest some degree of ciprofloxacin intercalation into duplex.
Assuntos
Ciprofloxacina/metabolismo , DNA de Cadeia Simples/metabolismo , DNA/metabolismo , Animais , Anti-Infecciosos/química , Anti-Infecciosos/metabolismo , Soluções Tampão , Ciprofloxacina/química , DNA/química , DNA de Cadeia Simples/química , Concentração de Íons de Hidrogênio , Estrutura Molecular , Oligonucleotídeos/metabolismo , Polinucleotídeos/metabolismo , Soluções , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta , Eletricidade Estática , TemperaturaRESUMO
RNA viruses have specific mutation rates that balance the conflicting needs of an evolutionary response to host antiviral defenses and avoidance of the error catastrophe. While most mutations are known to originate in replication errors, difficulties of capturing the underlying dynamics have left the mechanochemical basis of viral mutagenesis unresolved. Here, we use multiplexed magnetic tweezers to investigate error incorporation by the bacteriophage Φ6 RNA-dependent RNA polymerase. We extract large datasets fingerprinting real-time polymerase dynamics over four magnitudes in time, in the presence of nucleotide analogs, and under varying NTP and divalent cation concentrations and fork stability. Quantitative analysis reveals a new pause state that modulates polymerase fidelity and so ties viral polymerase pausing to the biological function of optimizing virulence. Adjusting the frequency of such pauses offers a target for therapeutics and may also reflect an evolutionary strategy for virus populations to track the gradual evolution of their hosts.
RESUMO
To develop improved methods of gene delivery, packaging DNA in chemical or viral vectors could increase electroporation-mediated transfection. To test this hypothesis, electroporation was applied to DU145 prostate cancer cells incubated with green fluorescent protein-encoded DNA plasmid either naked or packaged with cationic lipid (Lipofectin), polycationic peptide (salmon protamine) or retroviral vectors (Moloney murine leukemia viruses) and then assayed for gene expression and cell viability. Cationic lipid or electroporation alone each significantly increased transfection, but their combination was less effective. Addition of protamine peptide during electroporation was also less effective than electroporation alone. The combination of retroviral vectors and electroporation transfected fewer cells than retrovirus alone. We conclude that the combination of electroporation with chemical or viral vectors does not improve gene transfection in vitro.
Assuntos
Eletroporação/métodos , Terapia Genética/métodos , Lipídeos/genética , Peptídeos/genética , Transfecção/métodos , Vírus/genética , Animais , Sistemas de Liberação de Medicamentos/métodos , Humanos , Lipídeos/administração & dosagem , Masculino , Camundongos , Peptídeos/administração & dosagem , Células Tumorais CultivadasRESUMO
Recent studies have found methyl-6-adenosine in thousands of mammalian genes, and this modification is most pronounced near the beginning of the 3' UTR. We present a perspective on current work and new single-molecule sequencing methods for detecting RNA base modifications.
Assuntos
Adenosina/metabolismo , Epigenômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Processamento Pós-Transcricional do RNA , RNA/metabolismo , Regiões 3' não Traduzidas , Animais , Células HEK293 , Células Hep G2 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , TranscriptomaRESUMO
The past few years have seen the application of single-molecule force spectroscopy techniques to the study of topoisomerases. Magnetic tweezers are particularly suited to the study of topoisomerases due to their unique ability to exert precise and straightforward control of the supercoiled state of DNA. Here, we illustrate in a stepwise fashion how the dynamic properties of type IB topoisomerases can be monitored using this technique.
Assuntos
DNA Topoisomerases Tipo I/metabolismo , DNA , Magnetismo , Conformação de Ácido Nucleico , DNA/química , DNA/metabolismo , Quebras de DNA de Cadeia Simples , DNA Topoisomerases Tipo I/química , Humanos , Magnetismo/instrumentação , Magnetismo/métodos , Microscopia/instrumentação , Microscopia/métodos , SoftwareRESUMO
It is well known that multivalent cations cause free DNA in solution to condense into nanometer-scale particles with toroidal and rod-like morphologies. However, it has not been shown to what degree kinetic factors (e.g., condensate nucleation) versus thermodynamic factors (e.g., DNA bending energy) determine experimentally observed relative populations of toroids and rods. It is also not clear how multimolecular DNA toroids and rods interconvert in solution. We have conducted a series of condensation studies in which DNA condensate morphology statistics were measured as a function of time and DNA structure. Here, we show that in a typical in vitro DNA condensation reaction, the relative rod population 2 min after the initiation of condensation is substantially greater than that measured after morphological equilibrium is reached (ca. 20 min). This higher population of rods at earlier time points is consistent with theoretical studies that have suggested a favorable kinetic pathway for rod nucleation. By using static DNA loops to alter the kinetics and thermodynamics of condensation, we further demonstrate that reported increases in rod populations associated with decreasing DNA length are primarily due to a change in the thermodynamics of DNA condensation, rather than a change in the kinetics of condensate nucleation or growth. The results presented also reveal that the redistribution of DNA from rods to toroids is mediated through the exchange of DNA strands with solution.
Assuntos
DNA/química , DNA/ultraestrutura , Conformação de Ácido Nucleico , Sequência de Bases , DNA/metabolismo , Replicação do DNA , Indicadores e Reagentes , Cinética , Microscopia Eletrônica , Modelos Moleculares , Dados de Sequência Molecular , TermodinâmicaRESUMO
Toroidal DNA condensates have attracted the attention of biophysicists, biochemists, and polymer physicists for more than thirty years. In the biological community, the quest to understand DNA toroid formation has been motivated by its relevance to gene packing in certain viruses and by the potential use of DNA toroids in artificial gene delivery (e.g., gene therapy). In the physical sciences, DNA toroids are appreciated as a superb model system for studying particle formation by the collapse of a semiflexible, polyelectrolyte polymer. This review focuses on experimental studies from the past few years that have significantly increased our understanding of DNA toroid structure and the mechanism of their formation. Highlights include structural studies that show the DNA strands within toroids to be packed in an ideal hexagonal lattice, and also in regions with a nonhexagonal lattice that are required by the topological constraints associated with winding DNA into a toroid. Recent studies of DNA toroid formation have also revealed that toroid size limits result from a complex interplay between kinetic and thermodynamic factors that govern both toroid nucleation and growth. The work discussed in this review indicates that it will ultimately be possible to obtain substantial control over DNA toroid dimensions.
Assuntos
Biofísica/métodos , DNA/química , Cátions , Microscopia Crioeletrônica , DNA Circular , Cinética , Microscopia Eletrônica de Transmissão , Modelos Químicos , Conformação de Ácido Nucleico , Plasmídeos/metabolismo , Polímeros/química , TermodinâmicaRESUMO
The DNA of most vertebrate sperm cells is packaged by protamines. The primary structure of mammalian protamine I can be divided into three domains, a central DNA binding domain that is arginine-rich and amino- and carboxyl-terminal domains that are rich in cysteine residues. In native bull sperm chromatin, intramolecular disulfide bonds hold the terminal domains of bull protamine folded back onto the central DNA binding domain, whereas intermolecular disulfide bonds between DNA-bound protamines help stabilize the chromatin of mature mammalian sperm cells. Folded bull protamine was used to condense DNA in vitro under various solution conditions. Using transmission electron microscopy and light scattering, we show that bull protamine forms particles with DNA that are morphologically similar to the subunits of native bull sperm chromatin. In addition, the stability provided by intermolecular disulfide bonds formed between bull protamine molecules within in vitro DNA condensates is comparable with that observed for native bull sperm chromatin. The importance of the bull protamine terminal domains in controlling the bull sperm chromatin morphology is indicated by our observation that DNA condensates formed under identical conditions with a fish protamine, which lacks cysteine-rich terminal domains, do not produce as uniform structures as bull protamine. A model is also presented for the bull protamine.DNA complex in native sperm cell chromatin that provides an explanation for the positions of the cysteine residues in bull protamine that form intermolecular disulfide bonds.
Assuntos
Protaminas/química , Espermatozoides/metabolismo , Sequência de Aminoácidos , Animais , Arginina/química , Bovinos , Cromatina/química , Cromatina/metabolismo , Cisteína/química , DNA/química , DNA/metabolismo , Dissulfetos/química , Relação Dose-Resposta a Droga , Íons , Masculino , Mercaptoetanol/farmacologia , Microscopia Eletrônica , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Salmão , Fatores de TempoRESUMO
The process of DNA condensation into nanometer-scale particles has direct relevance to several fields, including cell biology, virology, and gene delivery for therapeutic purposes. DNA condensation has also attracted the attention of polymer physicists, as the collapse of DNA molecules from solution into well defined particles represents an exquisite example of a polymer phase transition. Here we present a quantitative study of DNA toroids formed by condensation of 3 kb DNA with hexammine cobalt (III). The presence or absence of static loops within this DNA molecule demonstrates the effect of nucleation loop size on toroid dimensions and that nucleation is principally decoupled from toroid growth. A comparison of DNA condensates formed at low ionic strength with those formed in the presence of additional salts (NaCl or MgCl2) shows that toroid thickness is a salt-dependant phenomenon. Together, these results have allowed the development of models for DNA toroid formation in which the size of the nucleation loop directly influences the diameter of the fully formed toroid, whereas solution conditions govern toroid thickness. The data presented illustrate the potential that exists for controlling DNA toroid dimensions. Furthermore, this study provides a set of data that should prove useful as a test for theoretical models of DNA condensation.