Full Width at Half Maximum of Nanopore Current Blockage Controlled by a Single-Biomolecule Interface

Abstract

A biological nanopore is one of the predominant singlemolecule approaches as a result of its controllable single-biomolecule interface, which could reflect the "intrinsic" information on an individual molecule in a label-free way. Because the current blockage is normally treated as the most important parameter for nanopore identification of every single molecule, the fluctuation of current blockage for certain types of molecules, defined as full width at half maximum (fwhm) of current blockage, actually owns a dominant influence on nanopore resolution. Therefore, controlling the fwhm of current blockage of molecules is critical for the sensing capability of the nanopore. Here, taking an aerolysin nanopore as a model, by precisely controlling the functional group in this single-biomolecule interface, we could narrow the fwhm of nanopore current blockage for DNA identification and prolong the duration inside the nanopore. Moreover, a substantial correlation between fwhm of current blockage and duration is established, showing a nonmonotonic variation. Besides, the mechanism is also clarified with studying the detailed current blockage events. This proposed correlation is further demonstrated to be applied uniformly across different mutant aerolysins for a certain DNA. This study proposes a new strategy for regulating molecular sensing from the duration of the analyte, which could guide the resolution of heterogeneity analysis using nanopores.

An engineered third electrostatic constriction of aerolysin to manipulate heterogeneously charged peptide transport

“…According to our previous studies, the R1 constricted region (T218 ∼ D222 and T274 ∼ S278) at the entrance of AeL determines the selectivity of the nanopore. 27–31 The introduction of charged residues at R1 achieved a charge selective AeL sensor. 32…”

Emerging Data Processing Methods for Single‐Entity Electrochemistry

“…Current traces (middle) and I b /I 0 histogram of the analytes (dA) 4 through WT (top), K238A (middle), and K238L (bottom) aeolysin nanopore. [35] Figures reproduced with permission from: a, ref. [36], Copyright 2020, Wiley-VCH; b, ref.…”

“…Copyright 2020, Wiley-VCH; b, ref [10],. Copyright 2022, Wiley-VCH; c, ref [35],. Copyright 2021, American Chemical Society.…”

Site‐Specific Introduction of Bioorthogonal Handles to Nanopores by Genetic Code Expansion

“…[8] The noncovalent pore-analyte interactions, governed by the side chains of 20 proteinogenic amino acids in the pore lumen, determine the molecular recognition events, and thus the resistive-pulse readout. To this end, chemical and biological strategies such as site-directed mutagenesis, [9] native chemical ligation, [10] internal adaptor installation, [6b, 11] SpyTag-SpyCatcher chemistry, [12] sulfhydryl chemistry, [13] and 2-cyanobenzothiazole (CBT) chemistry, [14] have been developed and used to facilitate nanopore engineering and functionalization. [15] The ideal scenario to manipulate the properties of nanopore building blocks without the limitation of amino acid site, type, number or reactivity however has not been achieved.…”