Energetics of base flipping at a DNA mismatch site confined at the latch constriction of α-hemolysin

Johnson, Robert P.; Perera, Rukshan T.; Fleming, Aaron M.; Burrows, Cynthia J.; White, Henry S.

doi:10.1039/c6fd00058d

Cited by 8 publications

(11 citation statements)

References 58 publications

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“…32–33 Here, we show that the kinetics of base flipping of a cytosine-cytosine pair situated at the latch constriction of αHL is significantly altered when one of the cytosine bases in the mismatch is modified at the carbon-5 position. Measuring the base flipping kinetics with a molecule-by-molecule approach, we are able to discriminate between duplexes containing a single mC, hmC, fC, or caC base.…”

Section: Introductionmentioning

confidence: 79%

Dynamics of a DNA Mismatch Site Held in Confinement Discriminate Epigenetic Modifications of Cytosine

et al. 2017

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The identification and discrimination of four epigenetic modifications to cytosine in the proposed active demethylation cycle is demonstrated at the single-molecule level, without the need for chemical pre-treatment or labeling. The wild-type protein nanopore α-hemolysin is used to capture individual DNA duplexes containing a single cytosine-cytosine mismatch. The mismatch is held at the latch constriction of α-hemolysin, which is used to monitor the kinetics of base flipping at the mismatch site. Base flipping and the subsequent interactions between the DNA and the protein are dramatically altered when one of the cytosine bases is replaced with methyl-, hydroxymethyl-, formyl-, or carboxylcytosine. As well as providing a route to single-molecule analysis of important epigenetic markers in DNA, our results provide important insights into how the introduction of biologically-relevant, but poorly understood, modifications to cytosine effect the local conformational dynamics of a DNA duplex in a confined environment.

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Section: Introductionmentioning

confidence: 79%

Dynamics of a DNA Mismatch Site Held in Confinement Discriminate Epigenetic Modifications of Cytosine

et al. 2017

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“…Hidden Markov Models (HMM) are used to quantify the primary signal traces. HMM have previously been successfully applied to timeseries data from single-molecule biophysics experiments (52)(53)(54)(55)(56). In the present case, the HMM assigns the "hidden" bound and unbound states of the sensor to segments of the observed signal trace that have statistically different current levels, typically with lower currents for the unbound state and higher currents for the bound state.…”

Section: Resultsmentioning

confidence: 99%

Molecular electronics sensors on a scalable semiconductor chip: A platform for single-molecule measurement of binding kinetics and enzyme activity

Fuller¹,

Padayatti²,

Abderrahim³

et al. 2022

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

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For nearly 50 years, the vision of using single molecules in circuits has been seen as providing the ultimate miniaturization of electronic chips. An advanced example of such a molecular electronics chip is presented here, with the important distinction that the molecular circuit elements play the role of general-purpose single-molecule sensors. The device consists of a semiconductor chip with a scalable array architecture. Each array element contains a synthetic molecular wire assembled to span nanoelectrodes in a current monitoring circuit. A central conjugation site is used to attach a single probe molecule that defines the target of the sensor. The chip digitizes the resulting picoamp-scale current-versus-time readout from each sensor element of the array at a rate of 1,000 frames per second. This provides detailed electrical signatures of the single-molecule interactions between the probe and targets present in a solution-phase test sample. This platform is used to measure the interaction kinetics of single molecules, without the use of labels, in a massively parallel fashion. To demonstrate broad applicability, examples are shown for probe molecule binding, including DNA oligos, aptamers, antibodies, and antigens, and the activity of enzymes relevant to diagnostics and sequencing, including a CRISPR/Cas enzyme binding a target DNA, and a DNA polymerase enzyme incorporating nucleotides as it copies a DNA template. All of these applications are accomplished with high sensitivity and resolution, on a manufacturable, scalable, all-electronic semiconductor chip device, thereby bringing the power of modern chips to these diverse areas of biosensing.

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“…The obtained activation energy for flipping a cytosine located at the latch constriction of α-HL is in good agreement with previously reported values for base flipping. 141 …”

Section: Biological Nanoporesmentioning

confidence: 99%

“…The obtained activation energy for flipping a cytosine located at the latch constriction of α-HL is in good agreement with previously reported values for base flipping. 141 Applications in Detection of Proteins. Direct detection of protein molecules with biological nanopores is challenging because the diameter of most biological nanopores, including α-HL, is smaller than the size of most protein molecules in folded and functional forms.…”

Section: ■ Biological Nanoporesmentioning

confidence: 99%

Nanopore Sensing

2016

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Energetics of base flipping at a DNA mismatch site confined at the latch constriction of α-hemolysin

Cited by 8 publications

References 58 publications

Dynamics of a DNA Mismatch Site Held in Confinement Discriminate Epigenetic Modifications of Cytosine

Dynamics of a DNA Mismatch Site Held in Confinement Discriminate Epigenetic Modifications of Cytosine

Molecular electronics sensors on a scalable semiconductor chip: A platform for single-molecule measurement of binding kinetics and enzyme activity

Nanopore Sensing

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