Ionic current blockades from DNA and RNA molecules in the alpha-hemolysin nanopore

We characterize the substate structure of current blockades produced when single-stranded polynucleotide molecules were electrophoretically driven into the alpha-hemolysin protein pore. We frequently observe substates where the ionic current is reduced by approximately 50%. Most of these substates can be associated with a molecular configuration where a polymer occupies only the vestibule region of the pore, though a few appear related to a polymer occupying only the transmembrane beta-barrel region of the pore. The duration of the vestibule configuration depends on polymer composition and on which end of the polymer, 3' or 5', subsequently threads into the narrowest constriction and initiates translocation. Below approximately 140 mV a polymer is more likely to escape from the vestibule against the applied voltage gradient, while at higher voltages a polymer is more likely to follow the voltage gradient by threading through the narrowest constriction and translocating through the pore. Increasing the applied voltage also increases the duration of the vestibule configuration. A semiquantitative model of these trends suggests that escape has stronger voltage dependence than threading, and that threading is sensitive to polymer orientation while escape is not. These results emphasize the utility of alpha-hemolysin as a model system to study biologically relevant physical and chemical processes at the single-molecule level.     

Microscopic Kinetics of DNA Translocation through synthetic nanopores

We have previously demonstrated that a nanometer-diameter pore in a nanometer-thick metal-oxide-semiconductor-compatible membrane can be used as a molecular sensor for detecting DNA. The prospects for using this type of device for sequencing DNA are avidly being pursued. The key attribute of the sensor is the electric field-induced (voltage-driven) translocation of the DNA molecule in an electrolytic solution across the membrane through the nanopore. To complement ongoing experimental studies developing such pores and measuring signals in response to the presence of DNA, we conducted molecular dynamics simulations of DNA translocation through the nanopore. A typical simulated system included a patch of a silicon nitride membrane dividing water solution of potassium chloride into two compartments connected by the nanopore. External electrical fields induced capturing of the DNA molecules by the pore from the solution and subsequent translocation. Molecular dynamics simulations suggest that 20-basepair segments of double-stranded DNA can transit a nanopore of 2.2 x 2.6 nm(2) cross section in a few microseconds at typical electrical fields. Hydrophobic interactions between DNA bases and the pore surface can slow down translocation of single-stranded DNA and might favor unzipping of double-stranded DNA inside the pore. DNA occluding the pore mouth blocks the electrolytic current through the pore; these current blockades were found to have the same magnitude as the blockade observed when DNA transits the pore. The feasibility of using molecular dynamics simulations to relate the level of the blocked ionic current to the sequence of DNA was investigated.     

DNA translocation governed by interactions with solid-state nanopores

We investigate the voltage-driven translocation dynamics of individual DNA molecules through solid-state nanopores in the diameter range 2.7-5 nm. Our studies reveal an order of magnitude increase in the translocation times when the pore diameter is decreased from 5 to 2.7 nm, and steep temperature dependence, nearly threefold larger than would be expected if the dynamics were governed by viscous drag. As previously predicted for an interaction-dominated translocation process, we observe exponential voltage dependence on translocation times. Mean translocation times scale with DNA length by two power laws: for short DNA molecules, in the range 150-3500 bp, we find an exponent of 1.40, whereas for longer molecules, an exponent of 2.28 dominates. Surprisingly, we find a transition in the fraction of ion current blocked by DNA, from a length-independent regime for short DNA molecules to a regime where the longer the DNA, the more current is blocked. Temperature dependence studies reveal that for increasing DNA lengths, additional interactions are responsible for the slower DNA dynamics. Our results can be rationalized by considering DNA/pore interactions as the predominant factor determining DNA translocation dynamics in small pores. These interactions markedly slow down the translocation rate, enabling higher temporal resolution than observed with larger pores. These findings shed light on the transport properties of DNA in small pores, relevant for future nanopore applications, such as DNA sequencing and genotyping.     

MspA nanopores from subunit dimers

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.     

Cloning of alpha-hemolysin gene from Staphylococcus aureus strain 015

The genomic library of Staphylococcus aureus O15 has been constructed on the EMBL-3 vector. The synthetic oligonucleotide probes to N- and C-end regions of alpha-hemolysin permitted identification of the recombinant bacteriophage clone RS-1 containing a gene for this protein. The restriction map of the cloned fragment has been constructed for restriction endonucleases SalGI, EcoRV, PstI, PvuII. Expression of the alpha-hemolysin gene in phagolysate of the recombinant clone RS-1 (1000 units per ml) has been demonstrated.     

Structural characterization of the alpha-hemolysin monomer from Staphylococcus aureus

Alpha-hemolysin from Staphylococcus aureus is secreted as a water-soluble monomer and assembles on membranes to oligomerize into a homo-heptameric, water-filled pore. These pores lead to lysis and cell death. Although the structure of the heptameric pore is solved by means of X-ray crystallography, structures of intermediate states-from the soluble monomer to all potential "pre-pore" structures-are yet unknown. Here, we propose a model of the monomeric alpha-hemolysin in solution based on molecular modeling, verified by small angle X-ray scattering data. This structure reveals details of the monomeric conformation of the alpha-hemolysin, for example inherent flexibility, along with definite differences in comparison to the structures used as templates.     

A parallel graph decomposition algorithm for DNA sequencing with nanopores

MOTIVATION: With the potential availability of nanopore devices that can sense the bases of translocating single-stranded DNA (ssDNA), it is likely that 'reads' of length approximately 10(5) will be available in large numbers and at high speed. We address the problem of complete DNA sequencing using such reads.We assume that approximately 10(2) copies of a DNA sequence are split into single strands that break into randomly sized pieces as they translocate the nanopore in arbitrary orientations. The nanopore senses and reports each individual base that passes through, but all information about orientation and complementarity of the ssDNA subsequences is lost. Random errors (both biological and transduction) in the reads create further complications. RESULTS: We have developed an algorithm that addresses these issues. It can be considered an extreme variation of the well-known Eulerian path approach. It searches over a space of de Bruijn graphs until it finds one in which (a) the impact of errors is eliminated and (b) both possible orientations of the two ssDNA sequences can be identified separately and unambiguously.Our algorithm is able to correctly reconstruct real DNA sequences of the order of 10(6) bases (e.g. the bacterium Mycoplasma pneumoniae) from simulated erroneous reads on a modest workstation in about 1 h. We describe, and give measured timings of, a parallel implementation of this algorithm on the Cray Multithreaded Architecture (MTA-2) supercomputer, whose architecture is ideally suited to this 'unstructured' problem. Our parallel implementation is crucial to the problem of rapidly sequencing long DNA sequences and also to the situation where multiple nanopores are used to obtain a high-bandwidth stream of reads.     

Ribosomal DNA in the grasshopper Podisma pedestris: escape from concerted evolution

Eukaryote nuclear ribosomal DNA (rDNA) typically exhibits strong concerted evolution: a pattern in which several hundred rDNA sequences within any one species show little or no genetic diversity, whereas the sequences of different species diverge. We report a markedly different pattern in the genome of the grasshopper Podisma pedestris. Single individuals contain several highly divergent ribosomal DNA groups. Analysis of the magnitude of divergence indicates that these groups have coexisted in the Podisma lineage for at least 11 million years. There are two putatively functional groups, each estimated to be at least 4 million years old, and several pseudogene groups, many of which are transcribed. Southern hybridization and real-time PCR experiments show that only one of the putatively functional types occurs at high copy number. However, this group is scarcely amplified under standard PCR conditions, which means that phylogenetic inference on the basis of standard PCR would be severely distorted. The analysis suggests that concerted evolution has been remarkably ineffective in P. pedestris. We propose that this outcome may be related to the species' exceptionally large genome and the associated low rate of deletion per base pair, which may allow pseudogenes to persist.     

Single-strand stacking free energy from DNA beacon kinetics

DNA beacons are short single-stranded chains which can form closed hairpin shapes through complementary base pairing at their ends. Contrary to the common polymer theory assumption that only their loop length matters, experiments show that their closing kinetics depend on the loop composition. We have modeled the closing kinetics and in so doing have obtained stacking enthalpies and entropies for single-stranded nucleic acids. The resulting change of persistence length with temperature effects the dynamics. With a Monte Carlo study, we answer another polymer question of how the closing time scales with chain length, finding tau approximately N(2.44+/-0.02). There is a significant crossover for shorter chains, bringing the effective exponent into good agreement with experiment.     

Purification of alpha-hemolysin from an overproducing E. coli strain

Summary The genetic determinant of the -hemolysin encoded by plasmid pHly152 has been cloned in both orientations in plasmid pBR322 giving rise to plasmids pSU157 and pSU158. E. coli strains carrying either of these recombinant Hly plasmids produced about 20 times more hemolysin activity than the parental plasmid pHly152, when grown in minimal medium supplemented with hemoglobin. Thus high hemolytic activity is not lethal to the cells, contrary to previous assumptions.-hemolysin was purified from culture supernatants of strain SU100 (pSU157) by ammonium sulfate precipitation and gel filtration in Sephacryl S-200 in the presence of 6 M urea. When purified -hemolysin preparations were subjected to electrophoretic analysis in denaturing conditions, a single 107 kdal polypeptide was observed. This probably corresponds to the -hemolysin protein, since an isogenic E. coli strain carrying plasmid pSU161, an Hly^- mutant derivative of pSU157, did not synthesize the 107 kdal polypeptide.     

DNA conformational kinetics

A model for the time dependence of DNA conformational state probabilities is formulated in the form of first-order differential equations. This model is applied to investigate the renaturation and denaturation rates for T2 and T7 DNA as reported in the series of experiments by Record and Zimm. Qualitative agreement is found in denaturation and for series of renaturation experiments with the same initial condition. However, partial agreement with series of renaturation experiments having the same final condition is obtained only by including an initial bimolecular step with properly matched pairs of strands. Comparison of all experiments with the calculated rates yields 5 × 10⁴ min^?1 as the step rate for melting a single base pair.     

Membrane insertion of Escherichia coli alpha-hemolysin is independent from membrane lysis

Escherichia coli alpha-hemolysin (HlyA) is a protein exotoxin that binds and lyses eukaryotic cell and model membranes in the presence of calcium. Previous studies have been able to distinguish between reversible toxin binding to the membrane and irreversible insertion into the lipid matrix. Membrane lysis occurs as the combined effect of protein insertion plus a transient perturbation of the membrane bilayer structure. In the past, insertion and bilayer perturbation have not been experimentally dissected. This has now been achieved by studying HlyA penetration into lipid monolayers at the air-water interface, in which three-dimensional effects (of the kind required to break down the bilayer permeability barrier) cannot occur. The study of native HlyA, together with the nonlytic precursor pro-HlyA, and of different mutants demonstrates that although some nonlytic variants (e.g. pro-HlyA) exhibit very low levels of insertion, others (e.g. the nonlytic mutant HlyA H859N) insert even more strongly than the lytic wild type. These results show that insertion does not necessarily lead to membrane lysis, i.e. that insertion and lysis are not "coupled" phenomena. Millimolar levels of Ca(2+), which are essential for the lytic activity, cause an extra degree of insertion but only in the case of the lytic forms of HlyA.     

Nonexponential Diffusion Extinction of Spin Echo from Water Protons in Wheat Root

The method of NMR spin echo with the magnetic field pulse gradient was used for studying self-diffusion of water molecules in the radial root direction. It was shown on the basis of physiologopharmacological methods of modification of the object that the coefficients of water self-diffusion resulting from computer decomposition of nonexponential diffusion extinction of the relative echo amplitude in the root to components are related to the subcellular and supercellular organization (structure) of the root and reflect changes in water permeability of the two transport channels of plasmodesmas.     

Rodent DNA reassociation kinetics

The kinetics of reassociation of the nuclear DNA of eight rodents has been examined to determine the amount and repetition frequency of various DNA fractions. Each rodent had at least two distinct, repeated DNA fractions, one group repeated about 300 times and the other repeated about 50,000 times. As shown, the actual repetition frequencies and the amount of the various DNA fractions varied from one species to another. Genetic implications of these results are discussed.