Nanopore Sequencing with MspA

Abstract

Nanopores may provide the basis for a high-speed and inexpensive de novo DNA sequencing technique that could revolutionize medical and biological science. In this technique, single-stranded DNA is electrophoretically translocated through a pore with inner dimensions similar to that of DNA. The co-passing ion current is recorded to obtain sequence information. Since its inception, nanopore sequencing has had promising results with only one bacterial pore α-Hemolysin and various solid-state pores. The geometry of another bacterial pore, MspA, found in the outer membrane of Mycobacteria Smegmatis, appears to be ideally suited for nanopore sequencing. We used site-directed mutagenesis on MspA to produce mutants that allow DNA translocation. These mutants can resolve small chains of the nucleotides A, C, and T when a duplex region of hairpin DNA arrests translocation. Additionally, DNA interaction with the mutant MspA is significantly and predictably altered with further mutations to the MspA structure. Our results introduce MspA as a promising and engineerable framework for nanopore sequencing technology.