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Issue 27, 2021
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Rapid and multiplex preparation of engineered Mycobacterium smegmatis porin A (MspA) nanopores for single molecule sensing and sequencing

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Abstract

Acknowledging its unique conical lumen structure, Mycobacterium smegmatis porin A (MspA) was the first type of nanopore that has successfully sequenced DNA. Recent developments of nanopore single molecule chemistry have also suggested MspA to be an optimum single molecule reactor. However, further investigations with this approach require heavy mutagenesis which is labor intensive and requires high end instruments for purifications. We here demonstrate an efficient and economic protocol which performs rapid and multiplex preparation of a variety of MspA mutants. The prepared MspA mutants were demonstrated in operations such as nanopore insertion, sequencing, optical single channel recording (oSCR), nanopore single molecule chemistry and nanopore rectification. The performance is no different from that of pores however prepared by other means. The time of all human operations and the cost for a single batch of preparation have been minimized to 40 min and 0.4$, respectively. This method is extremely useful in the screening of new MspA mutants, which has an urgent requirement in further investigations of new MspA nanoreactors. Its low cost and simplicity also enable efficient preparations of MspA nanopores for both industrial manufacturing and academic research.

Graphical abstract: Rapid and multiplex preparation of engineered Mycobacterium smegmatis porin A (MspA) nanopores for single molecule sensing and sequencing

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Supplementary files

Article information


Submitted
10 Mar 2021
Accepted
06 Jun 2021
First published
08 Jun 2021

This article is Open Access
All publication charges for this article have been paid for by the Royal Society of Chemistry

Chem. Sci., 2021,12, 9339-9346
Article type
Edge Article

Rapid and multiplex preparation of engineered Mycobacterium smegmatis porin A (MspA) nanopores for single molecule sensing and sequencing

S. Yan, L. Wang, X. Du, S. Zhang, S. Wang, J. Cao, J. Zhang, W. Jia, Y. Wang, P. Zhang, H. Chen and S. Huang, Chem. Sci., 2021, 12, 9339 DOI: 10.1039/D1SC01399H

This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. You can use material from this article in other publications without requesting further permissions from the RSC, provided that the correct acknowledgement is given.

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