Reading the primary sequence directly using nanopores remains challenging due to the complex building blocks of 20 proteinogenic amino acids and the corresponding sophisticated structures. Compared to the uniformly negatively charged polynucleotides, biological nanopores hardly provide effective ionic current responses to all heterogeneously charged peptides under nearly physiological pH conditions. Herein, we precisely design a N226Q/S228K mutant aerolysin which creates a new electrostatic constriction named R3 in-between two natural sensing regions for controlling the capture and translocation of heterogeneously charged peptides. At nearly physiological pH, the decoration of positive charges at this constriction gives a large velocity of electroosmotic flow (EOF), leading to a maximum 8-fold increase in frequency for the heterogeneously charged peptides with the net charge from +1 to -3. Even the duration time of the negatively charged peptide Aβ35-25D4 in N226Q/S228K AeL also rises from 0.07 ± 0.01 ms to 0.63 ± 0.01 ms after introducing the third electrostatic constriction. Therefore, the N226Q/S228K aerolysin nanopore with three electrostatic constrictions realizes the dual goals of both capturing and decelerating heterogeneously charged peptides without labelling, even for the folded peptides.
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