The electrophoretic passage of single-strand DNA through a nanopore has the potential to become an inexpensive, ultrafast DNA sequencing technique. All research in nanopore sequencing is based on the protein pore .-Hemolysin or involves artificial pores in inorganic materials. We propose to develop the Mycobacterium smegmatis porin A (MspA) into a new pore for nanopore sequencing. MspA is a promising platform for engineering a nanopore sequencing device for a number of reasons: (i) Its short, narrow constriction zone may give it higher sequencing sensitivity and resolution, (ii) MspA is extremely robust, (iii) Formation of stable MspA pores is easy and reliable, (iv) A wide range of stable MspA mutants can be readily engineered. In preliminary studies neither wild-type MspA nor MspA with a mutation in its constriction zone allowed translocation of DNA. Therefore, our goal is to tailor MspA for efficient translocation of DNA. We will remove excess negative charges from the rim and vestibule of the pore by site-directed mutagenesis, stabilize the loops near the constriction zone, and optimize the constriction zone for DNA passage. Translocation will be tested with a variety of ssDNA constructs in conditions designed to facilitate translocation. Once translocation is realized, further experiments will inform subsequent mutations to optimize MspA for nanopore sequencing. ? ? ? ?
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