title: A simple DNA handle attachment method for single molecule mechanical manipulation experiments.
abstract: Manipulating single molecules and systems of molecules with mechanical force is a powerful technique to examine their physical properties. Applying force requires attachment of the target molecule to larger objects using some sort of molecular tether, such as a strand of DNA. DNA handle attachment often requires difficult manipulations of the target molecule, which can preclude attachment to unstable, hard to obtain, and/or large, complex targets. Here we describe a method for covalent DNA handle attachment to proteins that simply requires the addition of a preprepared reagent to the protein and a short incubation. The handle attachment method developed here provides a facile approach for studying the biomechanics of biological systems.
authors.affiliation: Department of Chemistry and Biochemistry, UCLA-DOE Institute, Molecular Biology Institute, Los Angeles, California.
authors.forename: Duyoung
authors.initials: D
authors.lastname: Min
authors.affiliation: Department of Chemistry and Biochemistry, UCLA-DOE Institute, Molecular Biology Institute, Los Angeles, California.
authors.forename: Mark A
authors.initials: MA
authors.lastname: Arbing
authors.affiliation: Department of Chemistry and Biochemistry, UCLA-DOE Institute, Molecular Biology Institute, Los Angeles, California.
authors.forename: Robert E
authors.initials: RE
authors.lastname: Jefferson
authors.affiliation: Department of Chemistry and Biochemistry, UCLA-DOE Institute, Molecular Biology Institute, Los Angeles, California.
authors.forename: James U
authors.initials: JU
authors.lastname: Bowie
mesh_terms: D000595:Amino Acid Sequence; D001696:Biomechanical Phenomena; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004134:Dimyristoylphosphatidylcholine / Q000737:chemistry; D010450:Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D008051:Lipid Bilayers / Q000737:chemistry; D059346:Magnets; D058619:Maltose-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D052898:Optical Tweezers; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods*; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013194:Staining and Labeling / Q000379:methods*
chemical_list: D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C102898:GlpG protein, E coli; D008051:Lipid Bilayers; D058619:Maltose-Binding Proteins; D008565:Membrane Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins; D004247:DNA; D010450:Endopeptidases; D004134:Dimyristoylphosphatidylcholine
keywords: SpyCatcher; SpyTag; forced unfolding; magnetic tweezers; membrane protein; optical tweezers; protein folding