Single-molecule DNA detection with an engineered MspA protein nanopore

TLDR

Nanopores generate ionic current blockades that reveal target molecule identity, concentration, structure, and dynamics, and the Mycobacterium smegmatis porin MspA is exceptionally stable, modifiable, and features a short, narrow constriction ideal for sequencing short ssDNA segments. The study aims to engineer MspA to enable electronic detection and characterization of single ssDNA molecules. By removing negative charges in the channel constriction, the authors created an MspA mutant that can detect ssDNA as it is electrophoretically driven through the pore. A second mutant with positively charged vestibule residues increased interaction rates by ~20‑fold, reduced required voltage by half, and extended ssDNA residence by ~100‑fold, demonstrating MspA’s promise as an engineerable platform for nucleic acid analysis.

Abstract

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.