Nanopore Translocation Dynamics of a Single DNA-Bound Protein

Spiering, Andre; Getfert, Sebastian; Sischka, Andy; Reimann, Peter; Anselmetti, Dario

doi:10.1021/nl201541y

Cited by 52 publications

(75 citation statements)

References 53 publications

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“…These rely on the detection of a change in ionic current as a DNA molecule passes through a nanometer-sized hole and partially blocks it. Nanopores offer in principle straightforward operation, but the high speed of translocation makes it difficult to detect individual bound proteins, and the exerted force is ill defined [25].…”

Section: Discussionmentioning

confidence: 99%

Scanning a DNA Molecule for Bound Proteins Using Hybrid Magnetic and Optical Tweezers

et al. 2013

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The functional state of the genome is determined by its interactions with proteins that bind, modify, and move along the DNA. To determine the positions and binding strength of proteins localized on DNA we have developed a combined magnetic and optical tweezers apparatus that allows for both sensitive and label-free detection. A DNA loop, that acts as a scanning probe, is created by looping an optically trapped DNA tether around a DNA molecule that is held with magnetic tweezers. Upon scanning the loop along the λ-DNA molecule, EcoRI proteins were detected with ∼17 nm spatial resolution. An offset of 33±5 nm for the detected protein positions was found between back and forwards scans, corresponding to the size of the DNA loop and in agreement with theoretical estimates. At higher applied stretching forces, the scanning loop was able to remove bound proteins from the DNA, showing that the method is in principle also capable of measuring the binding strength of proteins to DNA with a force resolution of 0.1 pN/. The use of magnetic tweezers in this assay allows the facile preparation of many single-molecule tethers, which can be scanned one after the other, while it also allows for direct control of the supercoiling state of the DNA molecule, making it uniquely suitable to address the effects of torque on protein-DNA interactions.

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Section: Discussionmentioning

confidence: 99%

Scanning a DNA Molecule for Bound Proteins Using Hybrid Magnetic and Optical Tweezers

et al. 2013

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“…In 2006 Keyser et al [15] published the first experiment of optical trapping force measurement of DNA translocating through a nanopore giving a direct measurement of the electrical force exerted on DNA during translocation. This recent concept of coupling nanopores to optical tweezers opened up a variety of new single molecule studies for biomolecules [16,17]. This technique could be used to further explore molecular mechanics of transcription, to study forces applied by RNAP to DNA during transcription and give a more detailed view of the kinetic of transcription.…”

Section: Discussionmentioning

confidence: 99%

Detection of RNAP-DNA complexes using solid state nanopores

Raillon

Granjon

Gräf

et al. 2013

2013 35th Annual International Conference of the IEEE Engineering in Medicine and Biology Society (EMBC)

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Abstract-Transcription is the first step in gene expression where DNA is copied into RNA. It is extensively studied at the bulk level especially the regulation mechanism, which in cancerous cells is impaired. We were interested in studying E. coli RNAP enzyme at the single-molecule level for its functional as well as molecular motor properties. With nanopore sensing, we were able to observe RNA polymerase-DNA complexes translocate through nanopores and able to distinguish between individual complexes and bare RNA polymerase. We were also able to observe orientation of RNA polymerase in the nanopore whether flow or electric field predominates. The complexity of the signals from the protein-DNA complexes experiment motivated us to develop level detection software. This software is based on a change detection method called the CUSUM algorithm. OpenNanpore software was designed to analyze in details current blockages in nanopore signals with very little prior knowledge on the signal. With this work one can separate events according to their number of levels and study those subpopulations separately.

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“…The effective linear charge density of the DNA λ DN A includes both effects of counterion condensation and drag force from any electroosmotic flow 8,10,11 . The effects of electroosmotic flow are larger in nanocapillaries than in nanopores 9,12,13 .…”

Section: Free Energy Of the Systemmentioning

confidence: 99%

Single Molecule Localization and Discrimination of DNA–Protein Complexes by Controlled Translocation Through Nanocapillaries

et al. 2016

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Through the use of optical tweezers we performed controlled translocations of DNA–protein complexes through nanocapillaries. We used RNA polymerase (RNAP) with two binding sites on a 7.2 kbp DNA fragment and a dCas9 protein tailored to have five binding sites on λ-DNA (48.5 kbp). Measured localization of binding sites showed a shift from the expected positions on the DNA that we explained using both analytical fitting and a stochastic model. From the measured force versus stage curves we extracted the nonequilibrium work done during the translocation of a DNA–protein complex and used it to obtain an estimate of the effective charge of the complex. In combination with conductivity measurements, we provided a proof of concept for discrimination between different DNA–protein complexes simultaneous to the localization of their binding sites.

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Nanopore Translocation Dynamics of a Single DNA-Bound Protein

Cited by 52 publications

References 53 publications

Scanning a DNA Molecule for Bound Proteins Using Hybrid Magnetic and Optical Tweezers

Scanning a DNA Molecule for Bound Proteins Using Hybrid Magnetic and Optical Tweezers

Detection of RNAP-DNA complexes using solid state nanopores

Single Molecule Localization and Discrimination of DNA–Protein Complexes by Controlled Translocation Through Nanocapillaries

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