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 Ϸ20 higher interaction rates, required only half as much voltage to observe interaction, and allowed ssDNA to reside in the vestibule Ϸ100 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.DNA sequencing ͉ protein engineering ͉ bio-nanotechnology

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.bionanotechnology | next generation sequencing | single-molecule | stochastic sensing | protein pore T he information encoded in DNA is of paramount importance to medicine and to the life sciences. The mapping of the human genome is revolutionizing the understanding of genetic disorders and the prediction of disease and will aid in developing therapies as in refs. 1-3. The ability to sequence DNA quickly and inexpensively is essential to individualized medicine and to scientific research and has prompted the development of new sequencing techniques beyond the original Sanger sequencing (4-7). Nanopore DNA sequencing represents one of the approaches being developed to rapidly sequence a human genome for under $1,000 (www.genome.gov/12513210).In the most elementary form of nanopore DNA sequencing, a nanometer-scale pore provides the sole pathway for an ionic current. Single-stranded DNA (ssDNA) is electrophoretically driven through the pore, and as the ssDNA passes through, it reduces the ionic current through the pore. If each passing nucleotide yields a characteristic residual ionic current then the record of the current will correspond to the DNA sequence. This simple and reagent-free sequencing technique holds the promise to inexpensively read long lengths of DNA molecules at intrinsically fast rates (8). Due to its inherently small size, this system is amenable to parallelization (9).Lately, nanopore sequencing techniques have progressed substantially. This progress and the remaining challenges in nanopore DNA sequencing are summarized in a review article by Branton et al. (8). While nanotechnology usually involves materials such as Si and SiN, nanopore DNA sequencing first evolved using the well-studied protein porin α-hemolysin (10). In contrast to pores made from inorganic materials (11, 12), protein pores can be easily modified and produced with repeatable subnanometer accuracy. Stoddart (13) and Purnell (14) demonstrated that several locations within the beta barrel of α-hemolysin exhibit nucleotide-specific sensitivity with immobilized ssDNA (13). However, α-hemolysin's 5 nm-long cylindrical beta barrel presents...

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.

We investigate single-molecule electrophoretic translocation of A(50), C(50), A(25)C(50), and C(50)A(25) RNA molecules through the alpha-hemolysin transmembrane protein pore. We observe pronounced bilevel current blockages during translocation of A(25)C(50) and C(50)A(25) molecules. The two current levels observed during these bilevel blockages are very similar to the characteristic current levels observed during A(50) and C(50) translocation. From the temporal ordering of the two levels within the bilevel current blockages, we infer whether individual A(25)C(50) and C(50)A(25) molecules pass through the pore in a 3'-->5' or 5'-->3' orientation. Correlation between the level of current obstruction and the inferred A(25)C(50) or C(50)A(25) orientation indicates that 3'-->5' translocation of a poly C segment causes a significantly deeper current obstruction than 5'-->3' translocation. Our analysis also suggests that the 3' ends of C(50) and A(25)C(50) RNA molecules are more likely to initiate translocation than the 5' ends. Orientation dependent differences in a smaller current blockage that immediately precedes many translocation events suggest that this blockage also contains information about RNA orientation during translocation. These findings emphasize that the directionality of polynucleotide molecules is an important factor in translocation and demonstrate how structure within ionic current signals can give new insights into the translocation process.

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