DNA Garden: A Simple Method for Producing Arrays of Stretchable DNA for Single-Molecule Fluorescence Imaging of DNA-Binding Proteins

Abstract

Abstract We developed a simple method for producing arrays of stretchable DNAs, called DNA garden, for single-molecule fluorescence measurements. The method is based on microcontact printing of biotinylated bovine serum albumin (biotin-BSA) on a coverslip coated by 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer and on the subsequent tethering of neutravidin and biotinylated DNA. Without the need for a microfabricated substrate used for DNA tethering, it facilitates single-molecule investigations of DNA and DNA-binding proteins based on fluorescence microscopic imaging. The salient advantage of DNA garden is continuous observation of DNA in the repeated cycles of extension and relaxation by flow control, enabling the characterization of processes occurring in and on the relaxed DNA. The DNA garden was applied to the detection of cleavage sites of restriction enzymes and for the observation of the sliding dynamics of a tumor suppressor, p53, along extended DNA at the single-molecule level. Furthermore, experiments based on the repetitive extension and relaxation of DNA demonstrated that p53 causes looping of DNA, probably by connecting multiple regions of the relaxed DNA. The DNA garden is expected to be a powerful tool for the single-molecule imaging assay of DNA and DNA-binding proteins.