title: Scanning a DNA molecule for bound proteins using hybrid magnetic and optical tweezers.
abstract: The functional state of the genome is determined by its interactions with proteins that bind, modify, and move along the DNA. To determine the positions and binding strength of proteins localized on DNA we have developed a combined magnetic and optical tweezers apparatus that allows for both sensitive and label-free detection. A DNA loop, that acts as a scanning probe, is created by looping an optically trapped DNA tether around a DNA molecule that is held with magnetic tweezers. Upon scanning the loop along the λ-DNA molecule, EcoRI proteins were detected with ~17 nm spatial resolution. An offset of 33 ± 5 nm for the detected protein positions was found between back and forwards scans, corresponding to the size of the DNA loop and in agreement with theoretical estimates. At higher applied stretching forces, the scanning loop was able to remove bound proteins from the DNA, showing that the method is in principle also capable of measuring the binding strength of proteins to DNA with a force resolution of 0.1 pN/[Formula: see text]. The use of magnetic tweezers in this assay allows the facile preparation of many single-molecule tethers, which can be scanned one after the other, while it also allows for direct control of the supercoiling state of the DNA molecule, making it uniquely suitable to address the effects of torque on protein-DNA interactions.
authors.affiliation: Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Delft, The Netherlands.
authors.forename: Marijn T J
authors.initials: MT
authors.lastname: van Loenhout
authors.forename: Iwijn
authors.initials: I
authors.lastname: De Vlaminck
authors.forename: Benedetta
authors.initials: B
authors.lastname: Flebus
authors.forename: Johan F
authors.initials: JF
authors.lastname: den Blanken
authors.forename: Ludovit P
authors.initials: LP
authors.lastname: Zweifel
authors.forename: Koen M
authors.initials: KM
authors.lastname: Hooning
authors.forename: Jacob W J
authors.initials: JW
authors.lastname: Kerssemakers
authors.forename: Cees
authors.initials: C
authors.lastname: Dekker
mesh_terms: D010582:Bacteriophage lambda / Q000737:chemistry; D004279:DNA, Viral / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000032:analysis* / Q000737:chemistry; D015246:Deoxyribonuclease EcoRI / Q000032:analysis* / Q000737:chemistry; D060526:Magnetic Fields; D036103:Nanotechnology / Q000295:instrumentation* / Q000379:methods; D009690:Nucleic Acid Conformation; D052898:Optical Tweezers; D011485:Protein Binding; D019415:Torque
chemical_list: D004279:DNA, Viral; D004268:DNA-Binding Proteins; D015246:Deoxyribonuclease EcoRI