Preparation of DNA Nanostructures with Repetitive Binding Motifs by Rolling Circle Amplification

  • Edda Reiß
  • Ralph Hölzel
  • Frank F. Bier
Part of the Methods in Molecular Biology book series (MIMB, volume 749)


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.

Key words

Rolling circle amplification DNA nanostructure Single-stranded DNA Fluorescence microscopy SYBR Green II Phi29 DNA polymerase 



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).


  1. 1.
    Fire, A., and Xu, S. Q. (1995) Rolling replication of short DNA circles Proc. Natl. Acad. Sci. U. S. A. 92, 4641–5.Google Scholar
  2. 2.
    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.Google Scholar
  3. 3.
    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.Google Scholar
  4. 4.
    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.Google Scholar
  5. 5.
    Beyer, S., Nickels, P., and Simmel, F. C. (2005) Periodic DNA nanotemplates synthesized by rolling circle amplification Nano Lett. 5, 719–22.Google Scholar
  6. 6.
    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.Google Scholar
  7. 7.
    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.Google Scholar
  8. 8.
    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.Google Scholar
  9. 9.
    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.Google Scholar
  10. 10.
    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.Google Scholar
  11. 11.
    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.Google Scholar
  12. 12.
    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.Google Scholar
  13. 13.
    Epicentre Biotechnologies. Protocol for CircLigase™ ssDNA Ligase (continued Lit. #222) [homepage on the Internet]. No date [cited 2009 Jan 15]. Available from: Scholar
  14. 14.
    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.Google Scholar
  15. 15.
    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.Google Scholar
  16. 16.
    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.Google Scholar
  17. 17.
    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.Google Scholar
  18. 18.
    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.Google Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Department of Nanobiotechnology & NanomedicineFraunhofer Institute for Biomedical Engineering, Branch Potsdam-GolmPotsdamGermany

Personalised recommendations