Preparation of <i>Mycobacterium smegmatis</i> porin A (MspA) nanopores for single molecule sensing of nucleic acids

Yuqin Wang; Pingping Fan; Shanyu Zhang; Shuanghong Yan; Shuo Huang

doi:10.52601/bpr.2021.210016

Volume 7 Issue 5

Oct. 2021

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Article Navigation > Biophysics Reports > 2021 > 7(5): 355-364

Yuqin Wang, Pingping Fan, Shanyu Zhang, Shuanghong Yan, Shuo Huang. Preparation of Mycobacterium smegmatis porin A (MspA) nanopores for single molecule sensing of nucleic acids[J]. Biophysics Reports, 2021, 7(5): 355-364. doi: 10.52601/bpr.2021.210016

Citation:

Yuqin Wang, Pingping Fan, Shanyu Zhang, Shuanghong Yan, Shuo Huang. Preparation of Mycobacterium smegmatis porin A (MspA) nanopores for single molecule sensing of nucleic acids[J]. Biophysics Reports, 2021, 7(5): 355-364. doi: 10.52601/bpr.2021.210016

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Preparation of Mycobacterium smegmatis porin A (MspA) nanopores for single molecule sensing of nucleic acids

doi: 10.52601/bpr.2021.210016

Yuqin Wang^{1, 2, §},
Pingping Fan^{1, 2, §},
Shanyu Zhang^{1, 2},
Shuanghong Yan^{1, 2},
Shuo Huang^{1, 2
,
,}

1.
State Key Laboratory of Analytical Chemistry for Life Sciences, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023, China
2.
Chemistry and Biomedicine Innovation Center (ChemBIC), Nanjing University, Nanjing 210023, China

Funds: This project was funded by National Natural Science Foundation of China (31972917, 91753108 and 21675083), the Fundamental Research Funds for the Central Universities (020514380257 and 020514380261), programs for high-level entrepreneurial and innovative talents introduction of Jiangsu Province (individual and group program), Natural Science Foundation of Jiangsu Province (BK20200009), Excellent Research Program of Nanjing University (ZYJH004), Shanghai Municipal Science and Technology Major Project, State Key Laboratory of Analytical Chemistry for Life Science (5431ZZXM1902), Technology innovation fund program of Nanjing University, China Postdoctoral Science Foundation (2021M691508).

More Information

Corresponding author: shuo.huang@nju.edu.cn (S. Huang)
Received Date: 02 June 2021
Accepted Date: 25 June 2021
Publish Date: 31 October 2021

Abstract

Abstract

Mycobacterium smegmatis porin A (MspA), possessing a short (~0.6 nm) and narrow (~1.2 nm) constriction that enables its high spatial resolution of sensing, has emerged as an optimum choice for nanopore sequencing and nano-reactive sensing. However, prepared MspA nanopores cannot be obtained from any commercial vendors. We here provide a highly simplified protocol for MspA preparation. The protocol yields ~10 mg fully oligomerized protein per liter of culture and is straightforward to follow. With the prepared MspA nanopores, discrimination of immobilized ssDNA oligonucleotides can be performed, which serves as a demo application to demonstrate core procedures of single molecule sensing using MspA. This method is also in principle compatible with all other MspA mutants, which might merit the need to develop a variety of MspA based nano-reactive sensors.
- MspA nanopore,
- Prokaryotic expression,
- Affinity purification,
- Single molecule sensing

§Yuqin Wang and Pingping Fan contributed equally to this work.

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References(21)

References

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