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

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Laboratory of Nanoscale Biology, Institute of Bioengineering, School of Engineering, EPFL, 1015 Lausanne, Switzerland
Institute of Physics, Bijenička cesta 46, HR-10000 Zagreb, Croatia
§ Laboratory of Biological Network Characterization, Institute of Bioengineering, School of Engineering, EPFL, 1015 Lausanne, Switzerland
Cite this: Nano Lett. 2016, 16, 12, 7882–7890
Publication Date (Web):November 7, 2016
https://doi.org/10.1021/acs.nanolett.6b04165
Copyright © 2016 American Chemical Society

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    Abstract

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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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    • Analysis of force and current traces, localisation and work hysteresis, analytical solution for localisation shift, stochastic model details, equilibrium information from protein jump events (PDF)

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