Nanopore-Assisted, Sequence-Specific Detection, and Single-Molecule Hybridization Analysis of Short, Single-Stranded DNAs.
Anal Chem
; 91(13): 8630-8637, 2019 07 02.
Article
em En
| MEDLINE
| ID: mdl-31194518
ABSTRACT
We report here on the ability of the α-hemolysin (α-HL) nanopore to achieve label-free, selective, and real-time detection of 15 nt long ssDNA fragments in solution, by exploiting their hybridization with freely added, polycationic peptides-functionalized PNAs. At the core of our work lies the paradigm that when PNAs and ssDNA are mixed together, the bulk concentration of free PNA decreases, depending upon the (mis)match degree between complementary strands and their relative concentrations. We demonstrate that the ssDNA sensing principle and throughput of the method are determined by the rate at which nonhybridized, polycationic peptides-functionalized PNA molecules arrive at the α-HL's vestibule entrance and thread into the nanopore. We found that with the application of a 30-fold salt gradient across the nanopore, the method enhances single-molecule detection sensitivity in the nanomolar range of ssDNA concentrations. This study demonstrates that the transmembrane potential-dependent unzip of single PNA-DNA duplexes at the α-HL's ß-barrel entry permits discrimination between sequences that differ by one base pair.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
DNA de Cadeia Simples
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Técnicas Biossensoriais
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Ácidos Nucleicos Peptídicos
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Nanoporos
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Imagem Individual de Molécula
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Proteínas Hemolisinas
Tipo de estudo:
Diagnostic_studies
Limite:
Humans
Idioma:
En
Revista:
Anal Chem
Ano de publicação:
2019
Tipo de documento:
Article