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.
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References
Fire, A., and Xu, S. Q. (1995) Rolling replication of short DNA circles Proc. Natl. Acad. Sci. U. S. A. 92, 4641–5.
Liu, D., Daubendiek, S. L., Zillman, M. A., Ryan, K., and Kool, E. T. (1996) Rolling circle DNA synthesis: Small circular oligonucleotides as efficient templates for DNA polymerases J. Am. Chem. Soc. 118, 1587–94.
Lizardi, P. M., Huang, X., Zhu, Z., Bray-Ward, P., Thomas, D. C., and Ward, D. C. (1998) Mutation detection and single-molecule counting using isothermal rolling-circle amplification Nat. Genet. 19, 225–32.
Schweitzer, B., Roberts, S., Grimwade, B., Shao, W., Wang, M., Fu, Q., Shu, Q., Laroche, I., Zhou, Z., Tchernev, V. T., Christiansen, J., Velleca, M., and Kingsmore, S., F. (2002) Muliplexed protein profiling on microarrays by rolling-circle amplification Nat. Biotechnol. 20, 359–65.
Beyer, S., Nickels, P., and Simmel, F. C. (2005) Periodic DNA nanotemplates synthesized by rolling circle amplification Nano Lett. 5, 719–22.
Deng, Z., Tian, Y., Lee, S. H., Ribbe, A. E., and Mao, C. (2005) DNA-encoded self-assembly of gold nanoparticles into one-dimensional arrays Angew. Chem., Int. Ed. 44, 3582–5.
Zhao, W., Gao, Y., Kandadai, S. A., Brook, M. A., and Li, Y. (2006) DNA polymerization on gold nanoparticles through rolling circle amplification: Towards novel scaffolds for three-dimensional periodic nanoassemblies Angew. Chem., Int. Ed. 45, 2409–13.
Cheglakov, Z., Weizmann, Y., Braunschweig, A. B., Wilner, O. I., and Willner, I. (2008) Increasing the complexity of periodic protein nanostructures by the rolling-circle-amplified synthesis of aptamers Angew Chem., Int. Ed. 47, 126–30.
Zhao, W., Ali, M. M., Brook, M. A., and Li, Y. (2008) Rolling circle amplification: Applications in nanotechnology and biodetection with functional nucleic acids Angew. Chem., Int. Ed. 47, 6330–7.
Reiß, E., Hölzel, R., Nickisch-Rosenegk, M. v., and Bier, F. F. (2006) Rolling circle amplification for spatially directed synthesis of a solid phase anchored single-stranded DNA molecule, in DNA-Based Nanoscale Integration: International Symposium on DNA-Based Nanoscale Integration, Jena, Germany 18–20 May 2006 (Fritzsche, W., ed.) 2006, AIP Conference Proceedings 859, American Institute of Physics, Melville, NY, pp. 25–30.
Frieden, M., Pedroso, E., and Kool, E. T. (1999) Tightening the belt on polymerases: Evaluating the physical constraints on enzyme substrate size Angew. Chem., Int. Ed. 38, 3654–7.
Diegelman, A. M., and Kool, E. T. (2001) Chemical and enzymatic methods for preparing circular single-stranded DNAs Curr Protoc Nucleic Acid Chem Chapter 5, Unit 5.2.
Epicentre Biotechnologies. Protocol for CircLigase™ ssDNA Ligase (continued Lit. #222) [homepage on the Internet]. No date [cited 2009 Jan 15]. Available from: http://www.epibio.com/litindex.asp.
Blanco, L., Bernad, A., Lázaro, J. M., Martín, G., Garmendia, C., and Salas, M. (1989) Highly efficient DNA synthesis by the phage Phi 29 DNA polymerase. Symmetrical mode of DNA replication J. Biol. Chem. 264, 8935–40.
Reiß, E., Hölzel, R., and Bier, F. F. (2009) Synthesis and stretching of rolling circle amplification products in a flow-through system Small 5, 2316–22.
Feldkamp, U., Schroeder, H., and Niemeyer, C. M. (2006) Design and evaluation of single-stranded DNA carrier molecules for DNA-directed assembly J. Biomol. Struct. Dyn. 23, 657–66.
Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K., (Eds.) (1999) Short Protocols in Molecular Biology 4th ed., John Wiley & Sons, USA.
Dean, F. B., Nelson, J. R., Giesler, T. L., and Lasken, R. S. (2001) Rapid amplification of plasmid and phage DNA using Phi29 DNA polymerase and multiply-primed rolling circle amplification Genome Res. 11, 1095–9.
Acknowledgments
The authors thank the group of C.M. Niemeyer (University of Dortmund) for communicating DNA oligonucleotide sequence information. This work was supported by the European Union’s sixth framework program, contract no. NMP4-CT-2004-013775, under the project name NUCAN (Nucleic Acid Based Nanostructures).
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Reiß, E., Hölzel, R., Bier, F.F. (2011). Preparation of DNA Nanostructures with Repetitive Binding Motifs by Rolling Circle Amplification. In: Zuccheri, G., Samorì, B. (eds) DNA Nanotechnology. Methods in Molecular Biology, vol 749. Humana Press. https://doi.org/10.1007/978-1-61779-142-0_11
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DOI: https://doi.org/10.1007/978-1-61779-142-0_11
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