Construction and High Selectivity of Aerolysin Single Molecular Interface for Single Molecule Analysis †

doi:10.7503/cjcu20190364

Abstract

Abstract:

As an new conceptual single molecular interface, the biomolecular nanopore can accommodate only a single molecule and contains abundant amino acid residues in the lumen, providing an appropriate confinement for single molecule detection. Herein, a generalized method is proposed for the construct of single molecular interface based on membrane protein. The method is flexible, controllable and repeatable. The characterization of D209R mutation with Poly(dA)₄ illustrate that the selectivity of aerolysin single molecular interface is related the electrostatic properties at the entrance of nanopore, which deepens our understanding of aerolysin single molecular interface, providing a paradigm for rational design of functional single molecular interface.

Key words: Aerolysin, Single molecular interface, Nanopores, Single molecule analysis

CLC Number:

O657

TrendMD:

WU Xueyuan, YING Yilun, LONG Yitao. Construction and High Selectivity of Aerolysin Single Molecular Interface for Single Molecule Analysis ^†[J]. Chem. J. Chinese Universities, 2019, 40(9): 1825.

Figures/Tables 4

Fig.1 Construction of prokaryotic recombinant plasmid of pET-22b-Proaerolysin

Fig.2 Construction of aerolysin-based D209R single biomolecule interface (A) Illustration of the nanopore electrochemical measurement. Poly(dA)4 are used as the probe molecule to characterize the aerolysin-based single biomolecular interface. The location of 209 sites is marked by red band; (B) the chemical structure of negative-charge aspartic acid and positive-charge arginine; (C) the characteristic blockage signals of Poly(dA)4 in WT(black) and D209R(red) aerolysin single biomolecule interface.

Fig.3 Electrochemical characterization of aerolysin-based single biomolecule interface (A) SDS-PAGE result of WT and D209R mutant proaerolysin; (B) raw current traces of Poly(dA)4 in D209R single biomolecule interface for 8 h. Poly(dA)4 were added to the cis solution at 2 h after the nanopore formation.

Fig.4 Statistical analysis of Poly(dA)4 in WT and D209R aerolysin single molecular interface (A) I-V curves of WT(black) and D209R(red) aerolysin single molecular interface. I/I0 (B), duration time(C) and frequency(D) of Poly(dA)4 vs. applied voltage ranging from 80 to 180 mV in WT(black) and D209R(red) aerolysin single molecular interface.

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