title: Preparation of DNA nanostructures with repetitive binding motifs by rolling circle amplification.
abstract: A long one-dimensional single-stranded DNA (ssDNA) molecule with a periodic sequence motif is an attractive building block for DNA nanotechnology because it allows the positioning of oligonucleotide-labeled particles or molecules with high spatial resolution via molecular self-assembly simply by hybridization reactions. In vitro enzymatic isothermal rolling circle amplification (RCA) produces such long concatemeric ssDNA molecules. These are complementary in sequence to their circular template. In this chapter, the preparation of stretched and surface-attached RCA products at the single molecule level is described. The methods presented comprise the enzymatic circularization of a ssDNA oligonucleotide, the covalent coupling of amino-modified primers to carboxylated fluorescence beads, the preparation of a hydrophobic glass substrate, the RCA in a flow-through system, the postsynthetic staining and stretching of the RCA products as well as the microscopic observation of individual ssDNA molecules.
authors.affiliation: Department of Nanobiotechnology & Nanomedicine, Fraunhofer Institute for Biomedical Engineering, Branch Potsdam-Golm, Potsdam, Germany. edda.reiss@ibmt.fraunhofer.de
authors.forename: Edda
authors.initials: E
authors.lastname: Reiss
authors.forename: Ralph
authors.initials: R
authors.lastname: Hölzel
authors.forename: Frank F
authors.initials: FF
authors.lastname: Bier
mesh_terms: D017103:Bacillus Phages / Q000201:enzymology; D004270:DNA, Circular / Q000737:chemistry* / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000737:chemistry* / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D008856:Microscopy, Fluorescence; D049329:Nanostructures / Q000737:chemistry*; D036103:Nanotechnology / Q000379:methods*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D009693:Nucleic Acid Hybridization; D009930:Organic Chemicals
chemical_list: D004270:DNA, Circular; D004277:DNA, Single-Stranded; D009930:Organic Chemicals; C098798:SYBR Green II; D004259:DNA-Directed DNA Polymerase