Controlling DNA Fragments Translocation across Nanopores with the Synergic Use of Site-Directed Mutagenesis, pH-Dependent Charge Tuning, and Electroosmotic Flow.
ACS Appl Mater Interfaces
; 16(30): 40100-40110, 2024 Jul 31.
Article
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| MEDLINE
| ID: mdl-39038810
ABSTRACT
Biological and solid-state nanopores are at the core of transformative techniques and nanodevices, democratizing the examination of matter and biochemical reactions at the single-molecule level, with low cost, portability, and simplicity in operation. One of the crucial hurdles in such endeavors is the fast analyte translocation, which limits characterization, and a rich number of strategies have been explored over the years to overcome this. Here, by site-directed mutagenesis on the α-hemolysin protein nanopore (α-HL), sought to replace selected amino acids with glycine, electrostatic binding sites were induced on the nanopore's vestibule and constriction region and achieved in the most favorable case a 20-fold increase in the translocation time of short single-stranded DNA (ssDNA) at neutral pH, with respect to the wild-type (WT) nanopore. We demonstrated an efficient tool of controlling the ssDNA translocation time, via the interplay between the nanopore-ssDNA surface electrostatic interactions and electroosmotic flow, all mediated by the pH-dependent ionization of amino acids lining the nanopore's translocation pathway. Our data also reveal the nonmonotonic, pH-induced alteration of ssDNA average translocation time. Unlike mildly acidic conditions (pH â¼ 4.7), at a pH â¼ 2.8 maintained symmetrically or asymmetrically across the WT α-HL, we evidenced the manifestation of a dominant electroosmotic flow, determining the speeding up of the ssDNA translocation across the nanopore by counteracting the ssDNA-nanopore attractive electrostatic interactions. We envision potential applications of the presented approach by enabling easy-to-use, real-time detection of short ssDNA sequences, without the need for complex biochemical modifications to the nanopore to mitigate the fast translocation of such sequences.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
ADN de Cadena Simple
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Mutagénesis Sitio-Dirigida
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Electroósmosis
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Nanoporos
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Proteínas Hemolisinas
Idioma:
En
Revista:
ACS Appl Mater Interfaces
Asunto de la revista:
BIOTECNOLOGIA
/
ENGENHARIA BIOMEDICA
Año:
2024
Tipo del documento:
Article
País de afiliación:
Rumanía
Pais de publicación:
Estados Unidos