title: Single Molecule Localization and Discrimination of DNA-Protein Complexes by Controlled Translocation Through Nanocapillaries.
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.
authors.affiliation: Laboratory of Nanoscale Biology, Institute of Bioengineering, School of Engineering, EPFL , 1015 Lausanne, Switzerland.
authors.forename: Roman D
authors.initials: RD
authors.lastname: Bulushev
authors.affiliation: Institute of Physics , Bijenička cesta 46, HR-10000 Zagreb, Croatia.
authors.forename: Sanjin
authors.initials: S
authors.lastname: Marion
authors.affiliation: Laboratory of Biological Network Characterization, Institute of Bioengineering, School of Engineering, EPFL , 1015 Lausanne, Switzerland.
authors.forename: Ekaterina
authors.initials: E
authors.lastname: Petrova
authors.affiliation: Laboratory of Nanoscale Biology, Institute of Bioengineering, School of Engineering, EPFL , 1015 Lausanne, Switzerland.
authors.forename: Sebastian J
authors.initials: SJ
authors.lastname: Davis
authors.affiliation: Laboratory of Biological Network Characterization, Institute of Bioengineering, School of Engineering, EPFL , 1015 Lausanne, Switzerland.
authors.forename: Sebastian J
authors.initials: SJ
authors.lastname: Maerkl
authors.affiliation: Laboratory of Nanoscale Biology, Institute of Bioengineering, School of Engineering, EPFL , 1015 Lausanne, Switzerland.
authors.forename: Aleksandra
authors.initials: A
authors.lastname: Radenovic
mesh_terms: D001426:Bacterial Proteins / Q000032:analysis*; D001665:Binding Sites; D000076987:CRISPR-Associated Protein 9; D004247:DNA / Q000032:analysis*; D012321:DNA-Directed RNA Polymerases / Q000032:analysis*; D004720:Endonucleases / Q000032:analysis*; D043942:Nanotubes; D052898:Optical Tweezers
chemical_list: D001426:Bacterial Proteins; D004247:DNA; D012321:DNA-Directed RNA Polymerases; D000076987:CRISPR-Associated Protein 9; C000606107:Cas9 endonuclease Streptococcus pyogenes; D004720:Endonucleases
keywords: DNA−protein complex; Jarzynski equation; Nanopore; force measurements; nanocapillary; protein binding site