Stochastic Detection of Enantiomers

Kang, Xiaofeng; Cheley, Stephen; Guan, Xiyun; Bayley, Hagan

doi:10.1021/ja063485l

Cited by 154 publications

(145 citation statements)

References 34 publications

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“…Our results will be useful in several areas of basic science and biotechnology. By using host molecules lodged within the αHL pore, host-guest interactions can be investigated in fine detail at the single-molecule level (17,49). The present work will now permit us to examine binding events at a known face of a CD.…”

Section: Resultsmentioning

confidence: 96%

Molecular bases of cyclodextrin adapter interactions with engineered protein nanopores

Banerjee

Mikhailova

Cheley

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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106

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Engineered protein pores have several potential applications in biotechnology: as sensor elements in stochastic detection and ultrarapid DNA sequencing, as nanoreactors to observe singlemolecule chemistry, and in the construction of nano-and microdevices. One important class of pores contains molecular adapters, which provide internal binding sites for small molecules. Mutants of the α-hemolysin (αHL) pore that bind the adapter β-cyclodextrin (βCD) ∼10 4 times more tightly than the wild type have been obtained. We now use single-channel electrical recording, protein engineering including unnatural amino acid mutagenesis, and highresolution x-ray crystallography to provide definitive structural information on these engineered protein nanopores in unparalleled detail.alpha-hemolysin | single molecule | stochastic sensing | structure | unnatural amino acid M any research groups have used protein engineering to obtain enzymes and antibodies with new properties suited for specific tasks (1-6). Fewer groups have taken on the difficult problem of engineering membrane proteins (7). We have engineered the α-hemolysin protein pore, mindful of several potential applications in biotechnology, including its ability to act as a detector in stochastic sensing (8) and ultrarapid DNA sequencing (9), to serve as a nanoreactor for the observation of singlemolecule chemistry (10) and to act as a component for the construction of nano-and microdevices (11).An important breakthrough in this area, which enabled the stochastic sensing of organic molecules including the detection of DNA bases in the form of nucleoside monophosphates (12, 13), was the discovery of internal molecular adapters, a form of noncovalent protein modification (14). Most useful have been cyclodextrin (CD) adapters, which have until now been used in the absence of detailed structural information about how they work. The present paper is a definitive investigation, which provides such information through the application of a wide variety of technical approaches: single-channel electrical recording, protein engineering including unnatural amino acid mutagenesis, and x-ray crystallography. The studies employing mutagenesis show that the striking interactions seen in the crystal structures also occur in individual pores in lipid bilayers.We reveal that the tight-binding αHL mutants (15) M113N 7 and M113F 7 bind βCD in different orientations within the heptameric pore. In the case of M113N 7 , the top (primary hydroxyls) of the CD ring faces the trans entrance of the pore. In the case of M113F 7 , the bottom (secondary hydroxyls) of the CD ring faces the trans entrance, while the top of the ring is bonded to the pore through remarkable CH-π interactions. Another tight-binding mutant, M113V 7 , can bind the CD in both orientations. These results illustrate the exquisite level of engineering that can be achieved with protein nanopores, which is, for example, far beyond what is possible with solid-state pores. The work also provides information valuable for the design of ...

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Section: Resultsmentioning

confidence: 96%

Molecular bases of cyclodextrin adapter interactions with engineered protein nanopores

Banerjee

Mikhailova

Cheley

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

106

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“…If just one recognition site were to be used, it might be possible to sharpen R 2 by additional mutagenesis (with both natural and unnatural amino acids) and by site-directed chemical modification, and further blunt the other sites (48). Certainly, the exquisite ability of (context independent) stochastic sensing to distinguish between molecules (14,49) shows what can be done with nanopores under the most favorable conditions. In the case of solid-state nanopores, it has been suggested that an active property of translocating bases (such as the ability to carry tunneling currents) might be used for sequencing (10,(50)(51)(52), but no practical demonstration of such a technique has been made.…”

Section: Single-nucleotide Discrimination Within a Heteropolymeric Sementioning

confidence: 99%

Single-nucleotide discrimination in immobilized DNA oligonucleotides with a biological nanopore

Stoddart

Heron

Mikhailova

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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418

498

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The sequencing of individual DNA strands with nanopores is under investigation as a rapid, low-cost platform in which bases are identified in order as the DNA strand is transported through a pore under an electrical potential. Although the preparation of solid-state nanopores is improving, biological nanopores, such as ␣-hemolysin (␣HL), are advantageous because they can be precisely manipulated by genetic modification. Here, we show that the transmembrane ␤-barrel of an engineered ␣HL pore contains 3 recognition sites that can be used to identify all 4 DNA bases in an immobilized single-stranded DNA molecule, whether they are located in an otherwise homopolymeric DNA strand or in a heteropolymeric strand. The additional steps required to enable nanopore DNA sequencing are outlined.␣-hemolysin ͉ DNA sequencing ͉ genomics ͉ protein engineering ͉ protein pore

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“…3 Blocking currents enable the electrical identification of distinct molecular configurations and the elucidation of mechanisms responsible for single molecule behavior, such as single molecule transport, 4 protein unfolding, 5 and DNA unzipping. 6 Therefore, this method has been attracting attention as a next-generation of DNA sequencing or a single-molecule detection technique.…”

Section: Introductionmentioning

confidence: 99%

Hairpin DNA Unzipping Analysis Using a Biological Nanopore Array

2016

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This paper describes a method of hairpin DNA (hpDNA) unzipping analysis using a biological nanopore array. Various lengths of hpDNAs were captured and translocated through the nanopore and the individual duration times were monitored. The correlation between the unzipping time and the simulated hybridization energy of the duplexes was able to be modeled as first-ordered reaction. This method is one of the approach for understanding of basic biological reactions in DNA replication and RNA transcription.

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Stochastic Detection of Enantiomers

Cited by 154 publications

References 34 publications

Molecular bases of cyclodextrin adapter interactions with engineered protein nanopores

Molecular bases of cyclodextrin adapter interactions with engineered protein nanopores

Single-nucleotide discrimination in immobilized DNA oligonucleotides with a biological nanopore

Hairpin DNA Unzipping Analysis Using a Biological Nanopore Array

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