Clear Discrimination of Single-Molecule of a Single-Stranded DNA Homopolymers and Hetero-Homopolymers Through a New Mutant of Mycobacterium smegmatis Porin A, MspA

Biological nanopores have been proved as impressive nano-devices capable of holding the detection sensitivity from a nanoparticle to a single macromolecule. Protein pores are efficiently reproducible with assured channel geometry and their propensity to be engineered easily make them convenient in nanopore sensing techniques. Among various porin-proteins, MspA porin of Mycobacterium smegmatis is quite ideal for nanopore DNA sequencing. However, the traversing speed of singlestranded DNA (ssDNA) through both the mutants of MspA (M1MspA and M2MspA) is too fast to be analyzed. Therefore, we utilized a new mutant of MspA, termed as M3MspA which was previously purified in our laboratory. We, for the first time, demonstrated that translocation of ssDNA is achieved at a minimum voltage-threshold of 40 mV at pH 8.0. This new mutant of MspA exhibits a crystal-clear discrimination between two homopolymers, poly dA50 and poly dC50 with duration time up to few milliseconds and well-resolved signature current-amplitudes were obtained for both sides (3′ or 5′) of the polymer entered the pore, without any modification to ssDNA. Significantly, M3MspA determines a precise length of a hetero-homopolymer, poly(dA50dC50) by producing a dwell time congruent to the sum of the time spent by their component-homopolymers, separately. On account of their specificity and sensitivity towards translocating polynucleotides, we suggest that this new mutant of MspA would be a sophisticated bio-nanopore in further enlightening the translocation dynamics caused by diverse conformations of a same biomolecule. On top of that, it would also be helpful in nanopore DNA sequencing.