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Description
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The plasmid MspA-93M (D90N/D91N/D93M/D118R/D134R/E139K) is designed for the preparation of a mutant Mycobacterium smegmatis porin A (MspA) nanopore, which is engineered with a methionine at the site 93. It only has a single amino acid difference at the site 93 from the previously reported M2 MspA (D90N/D91N/D93N/D118R/D134R/E139K) (Proc. Natl. Acad. Sci., 2008, 105, 20647-20652), which has been applied in DNA sequencing (Nat. Biotechnol., 2012, 30, 349-353). MspA (PDB ID: 1UUN) possesses a narrow and short pore constriction, measuring ~1.2 nm at its narrowest spot and ~1.0 nm in an overall length. With this advantageous geometry, MspA exhibits an excellent sensing performance as a nanoreactor to probe single-molecule reactions (Nat. Commun., 2019, 10, 5668.; Chem. Sci., 2020,11, 879-887.; Anal. Chem. 2021, 93, 3, 1529–1536). The reported engineering of MspA to form corresponding nanoreactor has focused only on site 91. In our work, other potential engineering sites of MspA have been thoroughly investigated. The amino acid at the site 93 is located around the pore constriction and has a lumen-facing orientation. The MspA-93M mutant is designed to map the potential reactive sites within MspA to form a nanoreactor. The structure of the mutant, and its sensing performance to a model reactant (tetrachloroaurate(III) ion) are investigated. It is revealed that the sensing performance is inversely correlated to the cross-sectional diameter of the pore constriction size of the engineering site (ACS Sens. 2021. DOI: 10.1021/acssensors.1c00792.).
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