Precise Construction and Tuning of an Aerolysin Single-Biomolecule Interface for Single-Molecule Sensing

Wu, Xue-Yuan; Wang, Ming-Bo; Wang, Ya-Qian; Li, Meng-Yin; Ying, Yi-Lun; Huang, Jin; Long, Yi-Tao

doi:10.31635/ccschem.019.20180025

Cited by 13 publications

(9 citation statements)

References 45 publications

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“…The construction of recombinant proaerolysin plasmids refers to our previous work. − The primers used for plasmid mutation are as follows: T274S, 5′- GGTAGTACCAGTACCAGTCTGAGTCAGAGCG-3′ (forward) and 5′-CAGACTGGTACTGGTACTACCACCGTTTTG-3′ (reverse); T274V, 5′-GGTAGTACCGTCACCAGTCTGAGTCAGAGCG-3′ (forward) and 5′-CAGACTGGTGACGGTACTACCACCGTTTTG-3′ (reverse); T274W, 5′-GGTAGTACCTGGACCAGTCTGAGTCAGAGCG-3′ (forward) and 5′-CAGACTGGTCCAGGTACTACCACCGTTTTG-3′ (reverse), respectively. The mutant expression vectors were constructed by the site-directed mutagenesis with high-fidelity PCR enzymes (Invitrogen, 12368010).…”

Section: Experimental Methodsmentioning

confidence: 99%

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Revisiting the Origin of Nanopore Current Blockage for Volume Difference Sensing at the Atomic Level

Ying

et al. 2021

JACS Au

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Changes in the nanopore ionic current during entry of a target molecule underlie the sensing capability and dominate the intensity and extent of applications of the nanopore approach. The volume exclusion model has been proposed and corrected to describe the nanopore current blockage. However, increasing evidence shows nonconformity with this model, suggesting that the ionic current within a nanopore should be entirely reconsidered. Here, we revisit the origin of nanopore current blockage from a theoretical perspective and propose that the noncovalent interactions between a nanopore and a target molecule affect the conductance of the solution inside the nanopore, leading to enhanced current blockage. Moreover, by considering the example of an aerolysin nanopore discriminating the cytosine DNA and methylcytosine DNA that differ by a single methyl group, we completely demonstrate, by nanopore experiments and molecular dynamics simulations, the essential nature of this noncovalent interaction for discrimination. Our conductance model suggests multiplicative effects of both volume exclusion and noncovalent interaction on the current blockage and provides a new strategy to achieve volume difference sensing at the atomic level with highly specific current events, which would promote the nanopore protein sequencing and its applications in real-life systems.

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Section: Experimental Methodsmentioning

confidence: 99%

“…The mutant expression vectors were constructed by the site-directed mutagenesis with high-fidelity PCR enzymes (Invitrogen, 12368010). The PCR cycling conditions and the expression and purification processes of proaerolysin refer to our previous studies. − …”

Section: Experimental Methodsmentioning

confidence: 99%

Revisiting the Origin of Nanopore Current Blockage for Volume Difference Sensing at the Atomic Level

Ying

et al. 2021

JACS Au

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“…Aerolysin monomers are able to spontaneously oligomerize and insert into the lipid bilayer to form a nanopore after removal of the C-terminal peptide (CTP) by protease . Using the Ag/AgCl electrode to apply a transmembrane potential, the negatively charged DNA could be driven through the aerolysin nanopore, inducing the blockage in the ionic current.…”

Section: Resultsmentioning

confidence: 99%

“…Aerolysin monomers are able to spontaneously oligomerize and insert into the lipid bilayer to form a nanopore after removal of the C-terminal peptide (CTP) by protease. 35 Using the Ag/AgCl electrode to apply a transmembrane potential, the negatively charged DNA could be driven through the aerolysin nanopore, inducing the blockage in the ionic current. As illustrated in Figure 1A, the orange area defined as the R2 region containing positively charged K238 near the bottom of the lumen has been demonstrated to be critical for the translocation of oligonucleotides, 9,12,22,34 which is usually considered to be quite important for the nanopore to achieve high spatial resolution.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

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

Ying

et al. 2022

Langmuir

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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.

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Single-entity electrochemistry at confined sensing interfaces

et al. 2020

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Measurements at the single-entity level provide more precise diagnosis and understanding of basic biological and chemical processes. Recent advances in the chemical measurement provide a means for ultra-sensitive analysis. Confining the single analyte and electrons near the sensing interface can greatly enhance the sensitivity and selectivity. In this review, we summarize the recent progress in single-entity electrochemistry of single molecules, single particles, single cells and even brain analysis. The benefits of confining these entities to a compatible size sensing interface are exemplified. Finally, the opportunities and challenges of single entity electrochemistry are addressed.

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Precise Construction and Tuning of an Aerolysin Single-Biomolecule Interface for Single-Molecule Sensing

Cited by 13 publications

References 45 publications

Revisiting the Origin of Nanopore Current Blockage for Volume Difference Sensing at the Atomic Level

Revisiting the Origin of Nanopore Current Blockage for Volume Difference Sensing at the Atomic Level

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

Single-entity electrochemistry at confined sensing interfaces

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