Nanopore DNA sequencing with MspA.

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.

Single-molecule DNA detection with an engineered MspA protein nanopore.

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.

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.

Determination of RNA orientation during translocation through a biological nanopore.

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.