RESUMO
Fluorescence measurement is the main technology for post-amplification DNA detection in automated systems. Direct electrical reading of DNA concentration in solution could be an interesting alternative to go toward more miniaturized or less expensive devices, in particular in the pathogen detection field. Here we present the detection of short bacterial biomarkers with a direct impedancemetric measurement, within solutions of amplified and elongated DNA sequences in a microchannel. This technology relies on the electrohydrodynamic instability occurring in solutions of long charged macromolecules in a strong electric field. This instability specifically induces the aggregation of long DNAs and triggers conductivity variations that can be monitored by on-contact conductometry. An innovative isothermal amplification and elongation strategy was developed, combining SDA and HRCA reactions, in order to yield long DNAs suitable to be detected by the above principle, from a dilute initial DNA target. In contrast with previous label-free detection methods, this new strategy is very robust to matrix effects, thanks to the unique molecular weight dependence of the instability, coupled with this specific DNA amplification strategy. We demonstrate the detection of a 1 pM gene sequence specific to Staphylococcus aureus, in a portable system.
Assuntos
DNA Bacteriano/análise , Técnicas Eletroquímicas , Dispositivos Lab-On-A-Chip , Técnicas de Amplificação de Ácido Nucleico , Eletricidade , Hidrodinâmica , Staphylococcus aureusRESUMO
This article explores the role of some geometrical factors on the electrophoretically driven translocations of macromolecules through nanopores. In the case of asymmetric pores, we show how the entry requirements and the direction of translocation can modify the information content of the blocked ionic current as well as the transduction of the electrophoretic drive into a mechanical force. To address these effects we studied the translocation of single-stranded DNA through an asymmetric alpha-hemolysin pore. Depending on the direction of the translocation, we measure the capacity of the pore to discriminate between both DNA orientations. By unzipping DNA hairpins from both sides of the pores we show that the presence of single strand or double strand in the pore can be discriminated based on ionic current levels. We also show that the transduction of the electrophoretic drive into a denaturing mechanical force depends on the local geometry of the pore entrance. Eventually we discuss the application of this work to the measurement of energy barriers for DNA unzipping as well as for protein binding and unfolding.
Assuntos
Transporte Biológico/fisiologia , DNA de Cadeia Simples/fisiologia , DNA/fisiologia , Desnaturação de Ácido Nucleico/genética , DNA/química , DNA de Cadeia Simples/química , Nanoestruturas , Nanotecnologia , Conformação de Ácido Nucleico , PorosidadeRESUMO
We experimentally study the statistical distributions and the voltage dependence of the unzipping time of 45 base-pair-long double-stranded DNA through a nanopore. We then propose a quantitative theoretical description considering the nanopore unzipping process as a random walk of the opening fork through the DNA sequence energy landscape biased by a time-fluctuating force. To achieve quantitative agreement fluctuations need to be correlated over the millisecond range and have an amplitude of order k(B)T/bp. Significantly slower or faster fluctuations are not appropriate, suggesting that the unzipping process is efficiently enhanced by noise in the kHz range. We further show that the unzipping time of short 15 base-pair hairpins does not always increase with the global stability of the double helix and we theoretically study the role of DNA elasticity on the conversion of the electrical bias into a mechanical unzipping force.
Assuntos
DNA/química , DNA/ultraestrutura , Modelos Químicos , Modelos Moleculares , Simulação por Computador , Campos Eletromagnéticos , Modelos Estatísticos , Conformação de Ácido Nucleico , Desnaturação de Ácido Nucleico , Porosidade , Estresse MecânicoRESUMO
RNA polymerases carry out the synthesis of an RNA copy from a DNA template. They move along DNA, incorporate nucleotide triphosphate (NTP) at the end of the growing RNA chain, and consume chemical energy. In a single-molecule assay using the T7 RNA polymerase, we study how a mechanical force opposing the forward motion of the enzyme along DNA affects the translocation rate. We also study the influence of nucleotide and magnesium concentration on this process. The experiment shows that the opposing mechanical force is a competitive inhibitor of nucleotide binding. Also, the single-molecule data suggest that magnesium ions are involved in a step that does not depend on the external load force. These kinetic results associated with known biochemical and mutagenic data, along with the static information obtained from crystallographic structures, shape a very coherent view of the catalytic cycle of the enzyme: translocation does not take place upon NTP binding nor upon NTP cleavage, but rather occurs after PPi release and before the next nucleotide binding event. Furthermore, the energetic bias associated with the forward motion of the enzyme is close to kT and represents only a small fraction of the free energy of nucleotide incorporation and pyrophosphate hydrolysis.
Assuntos
Bacteriófago T7/enzimologia , Coenzimas/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Magnésio/metabolismo , Nucleotídeos/metabolismo , Proteínas Virais/metabolismo , Bacteriófago T7/genética , RNA Polimerases Dirigidas por DNA/genética , Transcrição Gênica , Proteínas Virais/genéticaRESUMO
We theoretically investigate the unzipping of DNA electrically driven through a nanometer-size pore. Taking the DNA base sequence explicitly into account, the unpairing and translocation process is described by a biased random walk in a one-dimensional energy landscape determined by the sequential basepair opening. Distributions of translocation times are numerically calculated as a function of applied voltage and temperature. We show that varying these two parameters changes the dynamics from a predominantly diffusive behavior to a dynamics governed by jumps over local energy barriers. The work suggests experimentally studying sequence effects, by comparing the average value and standard deviation of the statistical distribution of translocation times.
Assuntos
DNA/química , DNA/ultraestrutura , Eletroporação/métodos , Bicamadas Lipídicas/química , Modelos Químicos , Modelos Moleculares , Sequência de Bases , Simulação por Computador , DNA/efeitos da radiação , Bicamadas Lipídicas/efeitos da radiação , Dados de Sequência Molecular , Nanoestruturas/química , Nanoestruturas/efeitos da radiação , Nanoestruturas/ultraestrutura , Conformação de Ácido Nucleico , PorosidadeRESUMO
Electronic detection of the specific recognition between complementary DNA sequences is investigated. DNA probes are immobilized at different lateral positions on a Poly( L -lysine)-coated surface of an integrated silicon transistor array. Hybridization and field effect detection are done with the solid surface immersed in electrolyte solutions. Differential measurements are performed, where DNA hybridization leads to surface potential shifts between the transistors of the array. We experimentally show that these differential signals of hybridization can be enhanced significantly by changing the salt concentration between hybridization and detection.
Assuntos
Biofísica/métodos , DNA/química , Hibridização de Ácido Nucleico , Processamento de Sinais Assistido por Computador , Adsorção , Animais , Eletrólitos , Eletrônica , Elétrons , Técnicas Genéticas , Humanos , Conformação de Ácido Nucleico , Sais/farmacologiaRESUMO
The dc electrical conductivity of double stranded DNA is investigated experimentally. Single DNA molecules are manipulated with subpiconewton force and deposited on gold nanoelectrodes by optical traps. The DNA is modified at its ends for specific bead attachments and along the chain to favor charge transfer between the DNA base pair stack and the electrodes. For an electrode separation of 70 nm we find, in aqueous environment, electrical resistances above 100 G Omega indicating that even for weak stretching the double helix is almost insulating at this length scale.
Assuntos
DNA/química , Eletroquímica/métodos , Modelos Químicos , Água/química , Simulação por Computador , Elasticidade , Condutividade Elétrica , Estresse MecânicoRESUMO
A double-tweezer setup is used to induce mechanical stress in systems of molecular biology. A double strand of DNA is first stretched and the data is compared to precedent experiments to check the experimental setup. Then a short foldable fragment of RNA is probed; the typical unfolding/refolding hysteresis behaviour of this kind of construction is shown and followed by a study of its elasticity and a comparison to a worm-like chain model. Eventually, we describe the unfolding of a larger RNA structure, which unfolds by multiple steps. We show that this unfolding is not reversible and that it presents numerous unfolding pathways.
Assuntos
DNA/química , RNA/química , DNA/ultraestrutura , Escherichia coli/genética , Microscopia de Força Atômica/instrumentação , Microscopia de Força Atômica/métodos , Modelos Moleculares , Modelos Estruturais , Desnaturação de Ácido Nucleico , RNA/ultraestrutura , RNA Bacteriano/química , RNA Ribossômico/químicaRESUMO
An integrated array of field-effect transistor structures is used to detect two oppositely charged biopolymers: poly(L-lysine) and DNA. Local deposition of polymer solutions on part of the array induces sizeable variations in the dc current-voltage characteristics of the transistors exposed to the molecular charge. The whole transistor array is measured in the presence of a common electrolyte. Differential signals are studied as a function of electrolyte salt and polymer concentrations. The measurements provide information on the interface electrostatic potentials of the (semiconductor/biopolymer/electrolyte) system and the experimental data are compared to an analytical model which accounts for screening of the adsorbed charge by mobile ions.
Assuntos
Técnicas Biossensoriais/instrumentação , DNA/análise , Eletroquímica/instrumentação , Análise de Sequência com Séries de Oligonucleotídeos/instrumentação , Polilisina/análise , Transistores Eletrônicos , Biopolímeros/análise , Técnicas Biossensoriais/métodos , Eletroquímica/métodos , Eletrodos , Desenho de Equipamento , Análise de Falha de Equipamento , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
DNA is partly denatured in vitro by applying a force that mechanically separates the two strands of the double helix. Sudden reduction of the imposed displacement triggers spontaneous reannealing of the molecule. The corresponding force signals are measured by optical trapping interferometry for backward steps of various amplitudes and base sequence intervals. The measured signals frequently show plateaus of varying duration at discrete values that are dependent on the base sequence. Additional measurements are performed with proteins bound to the double helix. When the opening fork encounters such a protein during mechanical unzipping, force increases until the protein is ejected. This ejection induces fast release of tension and fast unzipping. Comparing our different measurements, we find that both DNA unzipping and the relaxation of tension in DNA are faster than the formation of the double helix.
Assuntos
DNA Viral/química , Desoxirribonucleases de Sítio Específico do Tipo II/química , Micromanipulação/métodos , Modelos Químicos , Modelos Moleculares , Simulação por Computador , DNA Viral/análise , Elasticidade , Movimento (Física) , Conformação de Ácido Nucleico , Desnaturação de Ácido Nucleico , Estimulação Física/métodos , Estresse MecânicoRESUMO
Force measurements are performed on single DNA molecules with an optical trapping interferometer that combines subpiconewton force resolution and millisecond time resolution. A molecular construction is prepared for mechanically unzipping several thousand-basepair DNA sequences in an in vitro configuration. The force signals corresponding to opening and closing the double helix at low velocity are studied experimentally and are compared to calculations assuming thermal equilibrium. We address the effect of the stiffness on the basepair sensitivity and consider fluctuations in the force signal. With respect to earlier work performed with soft microneedles, we obtain a very significant increase in basepair sensitivity: presently, sequence features appearing at a scale of 10 basepairs are observed. When measured with the optical trap the unzipping force exhibits characteristic flips between different values at specific positions that are determined by the base sequence. This behavior is attributed to bistabilities in the position of the opening fork; the force flips directly reflect transitions between different states involved in the time-averaging of the molecular system.
Assuntos
DNA/química , Interferometria/instrumentação , Interferometria/métodos , Lasers , Calibragem , Reação em Cadeia da Polimerase , Sensibilidade e EspecificidadeRESUMO
We describe the mechanical separation of the two complementary strands of a single molecule of bacteriophage lambda DNA. The 3' and 5' extremities on one end of the molecule are pulled progressively apart, and this leads to the opening of the double helix. The typical forces along the opening are in the range of 10-15 pN. The separation force signal is shown to be related to the local GC vs. AT content along the molecule. Variations of this content on a typical scale of 100-500 bases are presently detected.