title: High-throughput single-molecule analysis of DNA-protein interactions by tethered particle motion.
abstract: Tethered particle motion (TPM) monitors the variations in the effective length of a single DNA molecule by tracking the Brownian motion of a bead tethered to a support by the DNA molecule. Providing information about DNA conformations in real time, this technique enables a refined characterization of DNA-protein interactions. To increase the output of this powerful but time-consuming single-molecule assay, we have developed a biochip for the simultaneous acquisition of data from more than 500 single DNA molecules. The controlled positioning of individual DNA molecules is achieved by self-assembly on nanoscale arrays fabricated through a standard microcontact printing method. We demonstrate the capacity of our biochip to study biological processes by applying our method to explore the enzymatic activity of the T7 bacteriophage exonuclease. Our single molecule observations shed new light on its behaviour that had only been examined in bulk assays previously and, more specifically, on its processivity.
authors.affiliation: Centre National de la Recherche Scientifique, Institut de Pharmacologie et de Biologie Structurale, 205 route de Narbonne, Toulouse, F-31077, France.
authors.forename: Thomas
authors.initials: T
authors.lastname: Plénat
authors.forename: Catherine
authors.initials: C
authors.lastname: Tardin
authors.forename: Philippe
authors.initials: P
authors.lastname: Rousseau
authors.forename: Laurence
authors.initials: L
authors.lastname: Salomé
mesh_terms: D017123:Bacteriophage T7 / Q000201:enzymology; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005090:Exodeoxyribonucleases / Q000378:metabolism; D057166:High-Throughput Screening Assays; D046228:Microarray Analysis; D009038:Motion; D009690:Nucleic Acid Conformation; D020411:Oligonucleotide Array Sequence Analysis
chemical_list: D004268:DNA-Binding Proteins; D004247:DNA; D005090:Exodeoxyribonucleases