title: Disturbance-free rapid solution exchange for magnetic tweezers single-molecule studies.
abstract: Single-molecule manipulation technologies have been extensively applied to studies of the structures and interactions of DNA and proteins. An important aspect of such studies is to obtain the dynamics of interactions; however the initial binding is often difficult to obtain due to large mechanical perturbation during solution introduction. Here, we report a simple disturbance-free rapid solution exchange method for magnetic tweezers single-molecule manipulation experiments, which is achieved by tethering the molecules inside microwells (typical dimensions-diameter (D): 40-50 μm, height (H): 100 μm; H:D∼2:1). Our simulations and experiments show that the flow speed can be reduced by several orders of magnitude near the bottom of the microwells from that in the flow chamber, effectively eliminating the flow disturbance to molecules tethered in the microwells. We demonstrate a wide scope of applications of this method by measuring the force dependent DNA structural transitions in response to solution condition change, and polymerization dynamics of RecA on ssDNA/SSB-coated ssDNA/dsDNA of various tether lengths under constant forces, as well as the dynamics of vinculin binding to α-catenin at a constant force (< 5 pN) applied to the α-catenin protein.
authors.affiliation: Mechanobiology Institute, National University of Singapore, 117411, Singapore.
authors.forename: Shimin
authors.initials: S
authors.lastname: Le
authors.affiliation: Mechanobiology Institute, National University of Singapore, 117411, Singapore.
authors.forename: Mingxi
authors.initials: M
authors.lastname: Yao
authors.affiliation: Mechanobiology Institute, National University of Singapore, 117411, Singapore.
authors.forename: Jin
authors.initials: J
authors.lastname: Chen
authors.affiliation: Mechanobiology Institute, National University of Singapore, 117411, Singapore.
authors.forename: Artem K
authors.initials: AK
authors.lastname: Efremov
authors.affiliation: Department of Physics, National University of Singapore, 117542, Singapore.
authors.forename: Sara
authors.initials: S
authors.lastname: Azimi
authors.affiliation: Mechanobiology Institute, National University of Singapore, 117411, Singapore Department of Physics, National University of Singapore, 117542, Singapore Centre for Bioimaging Sciences, National University of Singapore, 117557, Singapore phyyj@nus.edu.sg.
authors.forename: Jie
authors.initials: J
authors.lastname: Yan
mesh_terms: D004247:DNA / Q000737:chemistry; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D046210:Microfluidic Analytical Techniques; D011938:Rec A Recombinases / Q000378:metabolism; D012965:Sodium Chloride; D016596:Vinculin / Q000378:metabolism; D051178:alpha Catenin / Q000378:metabolism
chemical_list: D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D051178:alpha Catenin; D016596:Vinculin; D012965:Sodium Chloride; D004247:DNA; D011938:Rec A Recombinases