.

Friday, November 18, 2016

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

Roman D. Bulushev, Sanjin Marion, Ekaterina Petrova, Sebastian J. Davis, Sebastian J. Maerkl, and Aleksandra Radenovic

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.

DOI
Post a Comment