Single-molecule DNA detection with an engineered MspA protein nanopore
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 remarkab...
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| Veröffentlicht in: | Proceedings of the National Academy of Sciences - PNAS 2008-12, Vol.105 (52), p.20647-20652 |
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| creator | Butler, Tom Z Pavlenok, Mikhail Derrington, Ian M Niederweis, Michael Gundlach, Jens H |
| description | 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 [almost equal to]20 higher interaction rates, required only half as much voltage to observe interaction, and allowed ssDNA to reside in the vestibule [almost equal to]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. |
| doi_str_mv | 10.1073/pnas.0807514106 |
| format | Article |
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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 [almost equal to]20 higher interaction rates, required only half as much voltage to observe interaction, and allowed ssDNA to reside in the vestibule [almost equal to]100 times longer than the first mutant. 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| subjects | Amino Acid Substitution Bacteria Bacterial Proteins - chemistry Bacterial Proteins - genetics Biochemistry Biological Sciences Biophysics Biosensing Techniques Crystallography, X-Ray Deoxyribonucleic acid DNA DNA probes DNA, Single-Stranded - analysis DNA, Single-Stranded - chemistry Electric current Electric potential Electrochemical Techniques Molecular probes Molecules Mutation, Missense Mycobacterium smegmatis Mycobacterium smegmatis - chemistry Mycobacterium smegmatis - genetics Nanotechnology Porins Porins - chemistry Porins - genetics Protein engineering Protein Structure, Quaternary - genetics Proteins Sequencing |
| title | Single-molecule DNA detection with an engineered MspA protein nanopore |
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