RESUMO
RNA polymerases (RNAPs) carry out the first step in the central dogma of molecular biology by transcribing DNA into RNA. Despite their importance, much about how RNAPs work remains unclear, in part because the small (3.4 Angstrom) and fast (~40 ms/nt) steps during transcription were difficult to resolve. Here, we used high-resolution nanopore tweezers to observe the motion of single Escherichia coli RNAP molecules as it transcribes DNA ~1,000 times improved temporal resolution, resolving single-nucleotide and fractional-nucleotide steps of individual RNAPs at saturating nucleoside triphosphate concentrations. We analyzed RNAP during processive transcription elongation and sequence-dependent pausing at the yrbL elemental pause sequence. Each time RNAP encounters the yrbL elemental pause sequence, it rapidly interconverts between five translocational states, residing predominantly in a half-translocated state. The kinetics and force-dependence of this half-translocated state indicate it is a functional intermediate between pre- and post-translocated states. Using structural and kinetics data, we show that, in the half-translocated and post-translocated states, sequence-specific protein-DNA interaction occurs between RNAP and a guanine base at the downstream end of the transcription bubble (core recognition element). Kinetic data show that this interaction stabilizes the half-translocated and post-translocated states relative to the pre-translocated state. We develop a kinetic model for RNAP at the yrbL pause and discuss this in the context of key structural features.
Assuntos
RNA Polimerases Dirigidas por DNA , Escherichia coli , Nanoporos , RNA Polimerases Dirigidas por DNA/metabolismo , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/genética , Escherichia coli/metabolismo , Escherichia coli/genética , Transcrição Gênica , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/química , Pinças Ópticas , Cinética , Nucleotídeos/metabolismoRESUMO
Nanopore DNA sequencing is limited by low base-calling accuracy. Improved base-calling accuracy has so far relied on specialized base-calling algorithms, different nanopores and motor enzymes, or biochemical methods to re-read DNA molecules. Two primary error modes hamper sequencing accuracy: enzyme mis-steps and sequences with indistinguishable signals. We vary the driving voltage from 100 to 200 mV, with a frequency of 200 Hz, across a Mycobacterium smegmatis porin A (MspA) nanopore, thus changing how the DNA strand moves through the nanopore. A DNA helicase moves the DNA through the nanopore in discrete steps, and the variable voltage moves the DNA continuously between these steps. The electronic signal produced with variable voltage is used to overcome the primary error modes in base calling. We found that single-passage de novo base-calling accuracy of 62.7 ± 0.5% with a constant driving voltage improves to 79.3 ± 0.3% with a variable driving voltage. The variable-voltage sequencing mode is complementary to other methods to boost the accuracy of nanopore sequencing and could be incorporated into any enzyme-actuated nanopore sequencing device.
Assuntos
DNA Helicases/genética , DNA/genética , Nanoporos , Porinas/genética , Algoritmos , DNA/isolamento & purificação , DNA Helicases/química , Mycobacterium smegmatis/genética , Porinas/química , Análise de Sequência de DNA/métodosRESUMO
Enzymes that operate on DNA or RNA perform the core functions of replication and expression in all of biology. To gain high-resolution access to the detailed mechanistic behavior of these enzymes, we developed single-molecule picometer-resolution nanopore tweezers (SPRNT), a single-molecule technique in which the motion of polynucleotides through an enzyme is measured by a nanopore. SPRNT reveals two mechanical substates of the ATP hydrolysis cycle of the superfamily 2 helicase Hel308 during translocation on single-stranded DNA (ssDNA). By analyzing these substates at millisecond resolution, we derive a detailed kinetic model for Hel308 translocation along ssDNA that sheds light on how superfamily 1 and 2 helicases turn ATP hydrolysis into motion along DNA. Surprisingly, we find that the DNA sequence within Hel308 affects the kinetics of helicase translocation.
Assuntos
DNA Helicases/metabolismo , Replicação do DNA/fisiologia , DNA de Cadeia Simples/química , Pinças Ópticas , Difosfato de Adenosina/química , Trifosfato de Adenosina/química , Humanos , Cinética , Imagem Individual de Molécula , Translocação Genética/fisiologiaRESUMO
Nanopore DNA sequencing is a promising single-molecule analysis technology. This technique relies on a DNA motor enzyme to control movement of DNA precisely through a nanopore. Specific experimental buffer conditions are required based on the preferred operating conditions of the DNA motor enzyme. While many DNA motor enzymes typically operate in salt concentrations under 100 mM, salt concentration simultaneously affects signal and noise magnitude as well as DNA capture rate in nanopore sequencing, limiting standard experimental conditions to salt concentrations greater than ~100 mM in order to maintain adequate resolution and experimental throughput. We evaluated the signal contribution from ions on both sides of the membrane (cis and trans) by varying cis and trans [KCl] independently during phi29 DNA Polymerase-controlled translocation of DNA through the biological porin MspA. Our studies reveal that during DNA translocation, the negatively charged DNA increases cation selectivity through MspA with the majority of current produced by the flow of K+ ions from trans to cis. Varying trans [K+] has dramatic effects on the signal magnitude, whereas changing cis [Cl-] produces only small effects. Good signal-to-noise can be maintained with cis [Cl-] as small as 20 mM, if the concentration of KCl on the trans side is kept high. These results demonstrate the potential of using salt-sensitive motor enzymes (helicases, polymerases, recombinases) in nanopore systems and offer a guide for selecting buffer conditions in future experiments to simultaneously optimize signal, throughput, and enzyme activity.
Assuntos
Proteínas de Bactérias/química , Porinas/química , Potássio/química , Cloretos/química , DNA de Cadeia Simples/química , Cinética , Nanotecnologia , Análise de Sequência de DNARESUMO
Single-molecule picometer resolution nanopore tweezers (SPRNT) is a new tool for analyzing the motion of nucleic acids through molecular motors. With SPRNT, individual enzymatic motions along DNA as small as 40pm can be resolved on sub-millisecond time scales. Additionally, SPRNT reveals an enzyme's exact location with respect to a DNA strand's nucleotide sequence, enabling identification of sequence-specific behaviors. SPRNT is enabled by a mutant version of the biological nanopore formed by Mycobacterium smegmatis porin A (MspA). SPRNT is strongly rooted in nanopore sequencing and therefore requires a solid understanding of basic principles of nanopore sequencing. Furthermore, SPRNT shares tools developed for nanopore sequencing and extends them to analysis of single-molecule kinetics. As such, this review begins with a brief history of our work developing the nanopore MspA for nanopore sequencing. We then describe the underlying principles of SPRNT, how it works in detail, and propose some potential future uses. We close with a comparison of SPRNT to other techniques and we present the methods that will enable others to use SPRNT.
Assuntos
Ácidos Nucleicos/química , Pinças Ópticas , Porinas/química , Imagem Individual de Molécula/métodos , Cinética , Mycobacterium smegmatis/enzimologia , Nanoporos/ultraestrutura , Ácidos Nucleicos/genética , Porinas/genéticaRESUMO
Malyshev et al. showed that the four-letter genetic code within a living organism could be expanded to include the unnatural DNA bases dNaM and d5SICS. However, verification and detection of these unnatural bases in DNA requires new sequencing techniques. Here we provide proof of concept detection of dNaM and d5SICS in DNA oligomers via nanopore sequencing using the nanopore MspA. We find that both phi29 DNA polymerase and Hel308 helicase are capable of controlling the motion of DNA containing dNaM and d5SICS through the pore and that single reads are sufficient to detect the presence and location of dNaM and d5SICS within single molecules.
Assuntos
DNA/análise , Desoxirribonucleotídeos/análise , Nanoporos , Nucleotídeos/análise , Porinas/genética , Fagos Bacilares , Proteínas de Bactérias/genética , DNA/genética , DNA Helicases/genética , DNA Polimerase Dirigida por DNA/genética , Desoxirribonucleotídeos/genética , Escherichia coli/genética , Código Genético , Íons , Bicamadas Lipídicas/química , Nucleotídeos/genética , Análise de Sequência de DNA , Thermococcus/metabolismoRESUMO
Techniques for measuring the motion of single motor proteins, such as FRET and optical tweezers, are limited to a resolution of â¼300 pm. We use ion current modulation through the protein nanopore MspA to observe translocation of helicase Hel308 on DNA with up to â¼40 pm sensitivity. This approach should be applicable to any protein that translocates on DNA or RNA, including helicases, polymerases, recombinases and DNA repair enzymes.
Assuntos
DNA Helicases/química , DNA/química , Micromanipulação/métodos , Proteínas Motores Moleculares/química , Nanoporos/ultraestrutura , DNA/ultraestrutura , DNA Helicases/ultraestrutura , Módulo de Elasticidade , Teste de Materiais/métodos , Proteínas Motores Moleculares/ultraestrutura , Movimento (Física) , Nanotecnologia/métodos , Ligação Proteica , Estresse MecânicoRESUMO
Nanopore sequencing of DNA is a single-molecule technique that may achieve long reads, low cost and high speed with minimal sample preparation and instrumentation. Here, we build on recent progress with respect to nanopore resolution and DNA control to interpret the procession of ion current levels observed during the translocation of DNA through the pore MspA. As approximately four nucleotides affect the ion current of each level, we measured the ion current corresponding to all 256 four-nucleotide combinations (quadromers). This quadromer map is highly predictive of ion current levels of previously unmeasured sequences derived from the bacteriophage phi X 174 genome. Furthermore, we show nanopore sequencing reads of phi X 174 up to 4,500 bases in length, which can be unambiguously aligned to the phi X 174 reference genome, and demonstrate proof-of-concept utility with respect to hybrid genome assembly and polymorphism detection. This work provides a foundation for nanopore sequencing of long, natural DNA strands.
Assuntos
DNA/genética , Nanoporos , Análise de Sequência de DNA/métodosRESUMO
Precise and efficient mapping of epigenetic markers on DNA may become an important clinical tool for prediction and identification of ailments. Methylated CpG sites are involved in gene expression and are biomarkers for diseases such as cancer. Here, we use the engineered biological protein pore Mycobacterium smegmatis porin A (MspA) to detect and map 5-methylcytosine and 5-hydroxymethylcytosine within single strands of DNA. In this unique single-molecule tool, a phi29 DNA polymerase draws ssDNA through the pore in single-nucleotide steps, and the ion current through the pore is recorded. Comparing current levels generated with DNA containing methylated CpG sites to current levels obtained with unmethylated copies of the DNA reveals the precise location of methylated CpG sites. Hydroxymethylation is distinct from methylation and can also be mapped. With a single read, the detection efficiency in a quasirandom DNA strand is 97.5 ± 0.7% for methylation and 97 ± 0.9% for hydroxymethylation.
Assuntos
5-Metilcitosina/metabolismo , Citosina/análogos & derivados , Metilação de DNA , Modelos Moleculares , Nanoporos , Porinas/metabolismo , 5-Metilcitosina/isolamento & purificação , Teorema de Bayes , Citosina/isolamento & purificação , Citosina/metabolismo , Epigenômica/métodos , Estrutura MolecularRESUMO
The protein nanopore Mycobacteria smegmatis porin A (MspA), can be used to sense individual nucleotides within DNA, potentially enabling a technique known as nanopore sequencing. In this technique, single-stranded DNA electrophoretically moves through the nanopore and results in an ionic current that is nucleotide-specific. However, with a high transport velocity of the DNA within the nanopore, the ionic current cannot be used to distinguish signals within noise. Through extensive (~100 µs in total) all-atom molecular dynamics simulations, we examine the effect of positively charged residues on DNA translocation rate and the ionic current blockades in MspA. Simulation of several arginine mutations show a ~10-30 fold reduction of DNA translocation speed without eliminating the nucleotide induced current blockages. Comparison of our results with similar engineering efforts on a different nanopore (α-hemolysin) reveals a nontrivial effect of nanopore geometry on the ionic current blockades in mutant nanopores.
Assuntos
Técnicas Biossensoriais/métodos , Condutometria/métodos , DNA/química , DNA/genética , Porinas/química , Porinas/genética , Análise de Sequência de DNA/métodos , Arginina/química , Arginina/genética , Sequência de Bases , Simulação por Computador , Condutividade Elétrica , Ativação do Canal Iônico , Modelos Químicos , Modelos Genéticos , Modelos Moleculares , Dados de Sequência Molecular , Mutação/genética , Translocação Genética/genéticaRESUMO
Mycobacterium smegmatis porin A (MspA) forms an octameric channel and represents the founding member of a new family of pore proteins. Control of subunit stoichiometry is important to tailor MspA for nanotechnological applications. In this study, two MspA monomers were connected by linkers ranging from 17 to 62 amino acids in length. The oligomeric pore proteins were purified from M. smegmatis and were shown to form functional channels in lipid bilayer experiments. These results indicated that the peptide linkers did not prohibit correct folding and localization of MspA. However, expression levels were reduced by 10-fold compared to wild-type MspA. MspA is ideal for nanopore sequencing due to its unique pore geometry and its robustness. To assess the usefulness of MspA made from dimeric subunits for DNA sequencing, we linked two M1-MspA monomers, whose constriction zones were modified to enable DNA translocation. Lipid bilayer experiments demonstrated that this construct also formed functional channels. Voltage gating of MspA pores made from M1 monomers and M1-M1 dimers was identical indicating similar structural and dynamic channel properties. Glucose uptake in M. smegmatis cells lacking porins was restored by expressing the dimeric mspA M1 gene indicating correct folding and localization of M1-M1 pores in their native membrane. Single-stranded DNA hairpins produced identical ionic current blockades in pores made from monomers and subunit dimers demonstrating that M1-M1 pores are suitable for DNA sequencing. This study provides the proof of principle that production of single-chain MspA pores in M. smegmatis is feasible and paves the way for generating MspA pores with altered stoichiometries. Subunit dimers enable better control of the chemical and physical properties of the constriction zone of MspA. This approach will be valuable both in understanding transport across the outer membrane in mycobacteria and in tailoring MspA for nanopore sequencing of DNA.
Assuntos
Nanoestruturas , Porinas/química , Dimerização , Ativação do Canal Iônico , Bicamadas Lipídicas , Modelos MolecularesRESUMO
Nanopore technologies are being developed for fast and direct sequencing of single DNA molecules through detection of ionic current modulations as DNA passes through a pore's constriction. Here we demonstrate the ability to resolve changes in current that correspond to a known DNA sequence by combining the high sensitivity of a mutated form of the protein pore Mycobacterium smegmatis porin A (MspA) with phi29 DNA polymerase (DNAP), which controls the rate of DNA translocation through the pore. As phi29 DNAP synthesizes DNA and functions like a motor to pull a single-stranded template through MspA, we observe well-resolved and reproducible ionic current levels with median durations of â¼28 ms and ionic current differences of up to 40 pA. Using six different DNA sequences with readable regions 42-53 nucleotides long, we record current traces that map to the known DNA sequences. With single-nucleotide resolution and DNA translocation control, this system integrates solutions to two long-standing hurdles to nanopore sequencing.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/instrumentação , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Nanoporos , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/genética , Nucleotídeos/química , Nucleotídeos/genética , Porinas/química , Porinas/genéticaRESUMO
Nanopore sequencing has the potential to become a fast and low-cost DNA sequencing platform. An ionic current passing through a small pore would directly map the sequence of single stranded DNA (ssDNA) driven through the constriction. The pore protein, MspA, derived from Mycobacterium smegmatis, has a short and narrow channel constriction ideally suited for nanopore sequencing. To study MspA's ability to resolve nucleotides, we held ssDNA within the pore using a biotin-NeutrAvidin complex. We show that homopolymers of adenine, cytosine, thymine, and guanine in MspA exhibit much larger current differences than in α-hemolysin. Additionally, methylated cytosine is distinguishable from unmethylated cytosine. We establish that single nucleotide substitutions within homopolymer ssDNA can be detected when held in MspA's constriction. Using genomic single nucleotide polymorphisms, we demonstrate that single nucleotides within random DNA can be identified. Our results indicate that MspA has high signal-to-noise ratio and the single nucleotide sensitivity desired for nanopore sequencing devices.
Assuntos
DNA/genética , Nanoporos , Nucleotídeos/genética , Porinas/metabolismo , Análise de Sequência de DNA/métodos , Citosina/metabolismo , Metilação de DNA , Eletricidade , Íons , Modelos Moleculares , Polimorfismo de Nucleotídeo Único/genéticaRESUMO
The cell wall of mycobacteria includes a thick, robust, and highly impermeable outer membrane made from long-chain mycolic acids. These outer membranes form a primary layer of protection for mycobacteria and directly contribute to the virulence of diseases such as tuberculosis and leprosy. We have formed in vitro planar membranes using pure mycolic acids on circular apertures 20 to 90 µm in diameter. We find these membranes to be long lived and highly resistant to irreversible electroporation, demonstrating their general strength. Insertion of the outer membrane channel MspA into the membranes was observed indicating that the artificial mycolic acid membranes are suitable for controlled studies of the mycobacterial outer membrane and can be used in nanopore DNA translocation experiments.
Assuntos
Lipídeos de Membrana/química , Membranas Artificiais , Mycobacterium tuberculosis/metabolismo , Ácidos Micólicos/química , 1,2-Dipalmitoilfosfatidilcolina/química , Permeabilidade da Membrana Celular , Concentração de Íons de Hidrogênio , Nanoporos , Porinas/químicaRESUMO
Nanopore sequencing has the potential to become a direct, fast, and inexpensive DNA sequencing technology. The simplest form of nanopore DNA sequencing utilizes the hypothesis that individual nucleotides of single-stranded DNA passing through a nanopore will uniquely modulate an ionic current flowing through the pore, allowing the record of the current to yield the DNA sequence. We demonstrate that the ionic current through the engineered Mycobacterium smegmatis porin A, MspA, has the ability to distinguish all four DNA nucleotides and resolve single-nucleotides in single-stranded DNA when double-stranded DNA temporarily holds the nucleotides in the pore constriction. Passing DNA with a series of double-stranded sections through MspA provides proof of principle of a simple DNA sequencing method using a nanopore. These findings highlight the importance of MspA in the future of nanopore sequencing.
Assuntos
DNA/análise , Mycobacterium smegmatis/química , Nanoestruturas/química , Porinas/metabolismo , Análise de Sequência de DNA/métodos , Sequência de Bases , DNA/química , DNA/metabolismo , Modelos Moleculares , Mycobacterium smegmatis/metabolismo , Conformação de Ácido Nucleico , Porosidade , Estrutura Terciária de ProteínaRESUMO
Nanopores hold great promise as single-molecule analytical devices and biophysical model systems because the ionic current blockades they produce contain information about the identity, concentration, structure, and dynamics of target molecules. The porin MspA of Mycobacterium smegmatis has remarkable stability against environmental stresses and can be rationally modified based on its crystal structure. Further, MspA has a short and narrow channel constriction that is promising for DNA sequencing because it may enable improved characterization of short segments of a ssDNA molecule that is threaded through the pore. By eliminating the negative charge in the channel constriction, we designed and constructed an MspA mutant capable of electronically detecting and characterizing single molecules of ssDNA as they are electrophoretically driven through the pore. A second mutant with additional exchanges of negatively-charged residues for positively-charged residues in the vestibule region exhibited a factor of approximately 20 higher interaction rates, required only half as much voltage to observe interaction, and allowed ssDNA to reside in the vestibule approximately 100 times longer than the first mutant. Our results introduce MspA as a nanopore for nucleic acid analysis and highlight its potential as an engineerable platform for single-molecule detection and characterization applications.