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Updated: Mar 10, 2026
Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions
Published on: August 27, 2014
Single Molecule Localization and Discrimination of DNA-Protein Complexes by Controlled Translocation Through
Roman D Bulushev1, Sanjin Marion2, Ekaterina Petrova3
1Laboratory of Nanoscale Biology, Institute of Bioengineering, School of Engineering, EPFL , 1015 Lausanne, Switzerland.
Abstract:
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.
