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Single-molecule DNA detection with an engineered MspA protein nanopore
Open accessAbstract
Nanopores hold great promise as single-molecule analytical devices and biophysical model systems because the ionic current blockades they produce contain information about the identity, concentration, structure, and dynamics of target molecules. The porin MspA of Mycobacterium smegmatis has remarkable stability against environmental stresses and can be rationally modified based on its crystal structure. Further, MspA has a short and narrow channel constriction that is promising for DNA sequencing because it may enable improved characterization of short segments of a ssDNA molecule that is threaded through the pore. By eliminating the negative charge in the channel constriction, we designed and constructed an MspA mutant capable of electronically detecting and characterizing single molecules of ssDNA as they are electrophoretically driven through the pore. A second mutant with additional exchanges of negatively-charged residues for positively-charged residues in the vestibule region exhibited a factor of ≈20 higher interaction rates, required only half as much voltage to observe interaction, and allowed ssDNA to reside in the vestibule ≈100 times longer than the first mutant. Our results introduce MspA as a nanopore for nucleic acid analysis and highlight its potential as an engineerable platform for single-molecule detection and characterization applications.
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Cited by
Three decades of nanopore sequencing
Deamer, D., Akeson, M., & Branton, D. (2016) · Nature Biotechnology · doi:10.1038/nbt.3423 · Cited by 1,292
Reading DNA at single-nucleotide resolution with a mutant MspA nanopore and phi29 DNA polymerase
Manrao, E. A., Derrington, I. M., Laszlo, A. H., Langford, K. W., Hopper, M. K., Gillgren, N., Pavlenok, M., Niederweis, M., & Gundlach, J. H. (2012) · Nature Biotechnology · doi:10.1038/nbt.2171 · Cited by 965
Self-citationSingle-nucleotide discrimination in immobilized DNA oligonucleotides with a biological nanopore
Stoddart, D., Heron, A. J., Mikhailova, E., Maglia, G., & Bayley, H. (2009) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.0901054106 · Cited by 482
Detection and mapping of 5-methylcytosine and 5-hydroxymethylcytosine with nanopore MspA
Laszlo, A. H., Derrington, I. M., Brinkerhoff, H., Langford, K. W., Nova, I. C., Samson, J. M., Bartlett, J. J., Pavlenok, M., & Gundlach, J. H. (2013) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1310240110 · Cited by 321
Self-citationTabatabaei, S. K., Pham, B., Pan, C., Liu, J., Chandak, S., Shorkey, S. A., Hernandez, A. G., Aksimentiev, A., Chen, M., Schroeder, C. M., et al. (2022) · Nano Letters · doi:10.1021/acs.nanolett.1c04203 · Cited by 63
Metadata
- Authors
- Butler, T. Z., Pavlenok, M., Derrington, I. M., Niederweis, M., & Gundlach, J. H.
- Venue
- Proceedings of the National Academy of Sciences
- Published
- 2008-12-30
- Volume
- 105
- Issue
- 52
- Pages
- 20647-20652
- ISSN
- 0027-8424 (ISSN-L)
- 1091-6490
- OpenAlex
- W2113231874
- Cited by
- 506
- References
- 0
- FWCI
- 11.09
- Type
- journal article
- Language
- en
- Open access
- green
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