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Published on: October 31, 2013
Single-molecule DNA detection with an engineered MspA protein nanopore
Tom Z Butler1, Mikhail Pavlenok, Ian M Derrington
1Department of Physics, University of Washington, Box 351560, 3910 15th Avenue NE, Seattle, WA 98195, USA.
Summary
Engineered bacterial porin MspA (Outer membrane protein A) enables electronic detection of single DNA molecules. Mutants show enhanced interaction and residence times for improved nanopore sequencing applications.
Area of Science:
- Biophysics
- Nanotechnology
- Molecular Biology
Background:
- Nanopores are promising for single-molecule analysis, providing data on molecular identity, concentration, structure, and dynamics via ionic current blockades.
- The Mycobacterium smegmatis porin MspA offers stability and structural tractability, with a narrow constriction suitable for DNA sequencing.
- MspA's potential for characterizing single-stranded DNA (ssDNA) relies on its ability to interact with and resolve short DNA segments.
Purpose of the Study:
- To engineer and construct MspA mutants for electronic detection and characterization of single ssDNA molecules.
- To investigate the impact of charge modifications in the MspA channel constriction and vestibule on ssDNA interaction.
- To establish MspA as a viable nanopore platform for nucleic acid analysis.
Main Methods:
- Rational design and construction of MspA mutants by altering charged residues within the pore.
- Electrophoretic manipulation of single ssDNA molecules through engineered MspA nanopores.
- Electronic detection and characterization of ssDNA translocation events by measuring ionic current blockades.
Main Results:
- An MspA mutant with a neutralized channel constriction successfully detected and characterized single ssDNA molecules.
- A second MspA mutant, with additional positive charges in the vestibule, demonstrated a ~20-fold increase in interaction rates.
- The second mutant required half the voltage for interaction and increased ssDNA residence time in the vestibule by ~100-fold.
Conclusions:
- Modified MspA porins serve as effective nanopores for single-molecule nucleic acid analysis.
- MspA is an engineerable platform with significant potential for single-molecule detection and characterization.
- These findings introduce MspA as a valuable tool for advancing nanopore-based sequencing and diagnostics.
