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Forschungsarbeit

DNA Origami as a Supramolecular Platform for Bionanotechnology

by Ralf Jungmann (31.07.2009)

Self-assembly using DNA molecules - which is based on their molecular recognition properties - offers the possibility to create complex nanostructures in a “bottom-up” approach. DNA origami is a method where a long, single stranded DNA molecule can be folded into arbitrary two-dimensional shapes using over 200 short oligonucleotides as staples [1]. The created structures are 100 nm in diameter and each oligonucleotide can be used as a 6 nm pixel to facilitate the precise arrangement of nanoparticles on an origami structure for example by means of DNA base pairing. We present a novel method for isothermal self-assembly of DNA structures using DNA origami as an example [2]. This method overcomes the need of heating up the solution to 95 °C and slowly cooling down. This should be of considerable importance for applications of the DNA origami technique where temperature-sensitive components, such as thiols or RNA, are to be used. In addition, we show the attachment of fluorescent quantum dots to origami structures using the specific binding of biotin to streptavidin. We are now exploring ways to detect fluorophores attached to origami structures using super-resolution fluorescence microscopy. We also work on combining single origami structures to form larger one- and two-dimensional assemblies. On the long run, we want to use this supramolecular platform to study physical, chemical and biological interactions at the nanometre scale, e.g. stochastic effects in the motion of molecular walkers on DNA origami or effects of spatially ordered multienzyme complexes which offer mechanistic advantages since reactions limited by the rate of diffusive transport are accelerated by the immediate proximity of the catalytic centers [3].

Formation of a rectangular DNA origami structure by thermal annealing[Bildunterschrift / Subline]: Figure 1: Formation of a rectangular DNA origami structure by thermal annealing.

In DNA origami, a long single stranded viral DNA molecule is folded using 226 short staple strands (each about 32 bases long). Staple and viral strands are mixed in a 100:1 ratio in folding buffer solution, heated to 95 °C and cooled slowly to form the desired structures (cp. Figure 1). Because each staple strand can serve as a 6 nm size pixel, the origami structures can be addressed with hairpin extended staple strands to “write“ arbitrary patterns on top of the origami (Figure 2A). This addressability to 6 nm can also be used to attach e.g. biomolecules to the origami structures. The staples are therefore labeled with biotin via a 4 Thymine bases long spacer (Figure 2B). Using the binding of streptavidin to biotin, streptavidin conjugated nanoparticles can be attached to these staples.

Addressing origami Structures with 6 nm precision. (A) A normal staple strand is replaced by one with a dumbbell hairpin structure for AFM height contrast. (B) Strategy for attaching streptavidin conjugated quantum dots to biotinylated staple strands.[Bildunterschrift / Subline]: Figure 2: Addressing origami Structures with 6 nm precision. (A) A normal staple strand is replaced by one with a dumbbell hairpin structure for AFM height contrast. (B) Strategy for attaching streptavidin conjugated quantum dots to biotinylated staple strands.

The denaturing agent formamide lowers DNA melting temperatures linearly by approximately 0.6 °C per % formamide in the buffer. The phenomenological equivalence between formamide concentration and temperature led us to investigate whether DNA nanostructures like origami can be assembled in a formamide-containing buffer, in which the formamide concentration is slowly lowered rather than by using a typical temperature based annealing protocol (cp. Figure 3).

Isothermal formation of DNA origami by dialysis over several stages against buffer solutions with successively decreasing formamide concentrations [2][Bildunterschrift / Subline]: Figure 3: Isothermal formation of DNA origami by dialysis over several stages against buffer solutions with successively decreasing formamide concentrations [2].

After self-assembly of the origami structures using thermal or isothermal annealing, a 5 µl drop (containing billions of structures) of the solution is deposited onto a freshly cleaved mica surface and imaged readily using an Atomic Force Microscopy (AFM) in fluid (Figure 4).

Rectangular DNA origami structures imaged in tapping mode under TAE/Mg2+ buffer. The origami structures in (A) were obtained by thermal annealing. The structures in (B) were prepared using the isothermal assembly method with formamide. Scale bars are 100 n[Bildunterschrift / Subline]: Figure 4: Rectangular DNA origami structures imaged in tapping mode under TAE/Mg2+ buffer. The origami structures in (A) were obtained by thermal annealing. The structures in (B) were prepared using the isothermal assembly method with formamide. Scale bars are 100 nm, height scale is 5 nm. (Imaged using a Nanoscope III AFM, Veeco Instruments).
Single hairpin structures are clearly resolved. The letter size is 60000 times smaller than font sizes used in a conventional newspaper. Scale in (A) is 100 nm, scale in (B) is 50 nm, height scale is 5 nm. (Imaged using a Nanowizard II AFM, courtesy of JPK[Bildunterschrift / Subline]: Figure 5: Single hairpin structures are clearly resolved. The letter size is 60000 times smaller than font sizes used in a conventional newspaper. Scale in (A) is 100 nm, scale in (B) is 50 nm, height scale is 5 nm. (Imaged using a Nanowizard II AFM, courtesy of JPK Instruments).

54 and 42 out of the 226 staple strands have been replaced by hairpin strands to resemble the TUM and NIM logo respectively (Figure 5).

Attaching fluorescent nanocrystals to DNA Origami structures.[Bildunterschrift / Subline]: Figure 6: Attaching fluorescent nanocrystals to DNA Origami structures. (A) AFM height image in fluid of streptavidin coated quantum dots attached to a biotinylated staple in a DNA Origami structure. (B) AFM TappingMode amplitude image with a cross section of the highlighted origami. The measured quantum dot conjugate diameter is 18 nm (specified diameter range: 15-20 nm). Length scales are 200 nm, height scale is 10 nm in (A) and 30 mV in (B). Insets are 180 nm x 160 nm.

A staple strand in the center of the rectangular origami structure was extended with a biotin label. After annealing, the origami structures were mixed in a 1:1 ratio with streptavidin conjugated quantum dots. AFM results are shown in Figure 6A together with a section analysis in Figure 6B. The measured diameter lies well in the range specified by the manufacturer.

Our future research will be focused on optically detecting fluorescent molecules on DNA origami structures using Total Internal Reflection Fluorescence Microscopy (TIRFM) and Super-Resolution Fluroescence Microscopy. Also spatial proximity of catalytic reaction centers are going to be investigated by attaching different molecules on top of an origami structure in a cascade-like geometry. Immediate proximity of these reaction centers is known to speed up enzymatic reactions that are normally limited by diffusive transport [3]. There are numerous examples in nature where cascades are found in spatial proximity, e.g. signal transduction or photosynthesis. This design would lead to a locally increased concentration of reactants and different reaction kinetics compared to experiments in bulk.

References:

[1] Rothemund, P., Folding DNA to create nanoscale shapes and patterns. Nature, 2006. 440(7082): p. 297-302.

[2] Jungmann, R., T. Liedl, T.L. Sobey, W. Shih, and F. Simmel, Isothermal assembly of DNA origami structures using denaturing agents. Journal Of The American Chemical Society, 2008. 130(31): p. 10062-10063.

[3] Niemeyer, C.M., J. Koehler, and C. Wuerdemann, DNA-directed assembly of bienzymic complexes from in vivo biotinylated NAD(P)H : FMN oxidoreductase and luciferase. ChemBioChem, 2002. 3(2-3): p. 242-245.


Education
  • 10/2001-10/2006
  • Studies in physics at Saarland University
  • 10/2005-10/2006
  • Diploma Thesis "Direct Micro- and Nanoscale Failure Visualization in the Nanocomposite Bone" at the University of California in Santa Barbara in the group of Prof. Dr. Paul K. Hansma
  • 11/2006 - 02/2007
  • Researcher at the Institute for Experimental Physics at Saarland University in the group of Prof. Dr. Karin Jacobs (Soft Matter Physics) and Prof. Dr. Christian Wagner (Nonlinear Dynamics and Pattern Formation)
  • 03/2007_11/2007
  • PhD student at LMU Munich, group of Prof. Dr. Jörg P. Kotthaus, AG Simmel
  • 11/2007-present
  • PhD student at TU Munich, group of Prof. Dr. Friedrich C. Simmel
  • 03/2008–present
  • Member of IDK CompInt

Publications
  • Journal publications
  • R. Jungmann, T. Liedl, T. L. Sobey, W. Shih, and F. C. Simmel, Isothermal assembly of DNA origami structures using denaturing agents, Journal of the American Chemical Society, 130, 10062-10063 (2008).
  • R. Jungmann, S. Renner, F. C. Simmel, From DNA nanotechnology to synthetic biology, HFSP Journal 2, 99-109 (2008).
  • Hansma, P., P. Turner, B. Drake, E. Yurtsev, A. Proctor, P. Mathews, J. Lelujian, C. Randall, J. Adams, R. Jungmann et al, The bone diagnostic instrument II: Indentation distance increase. Review of Scientific Instruments, 2008. 79(6): p. 064303-8.
  • Lauer, M.E., R. Jungmann, J.H. Kindt, S. Magonov, J.H. Fuhrhop, E. Oroudjev, and H.G. Hansma, Formation and Healing of Micrometer-Sized Channel Networks on Highly Mobile Au(111) Surfaces. Langmuir, 2007. 23(10): p. 5459-5465.
  • Thurner, P.J., B. Erickson, R. Jungmann et al, High-speed photography of compressed human trabecular bone correlates whitening to microscopic damage. Engineering Fracture Mechanics, 2007. 74(12): p. 1928-1941.
  • Book articles
  • P.J. Thurner, E. Oroudjev, R. Jungmann et al, Imaging of Bone Ultrastructure using Atomic Force Microscopy in: Modern Research and Educational Topics in Microscopy 3rd Edition, Vol.1, A. Méndez-Vilas and J. Díaz (Editors), Formatex: Badajoz, Spain (2007).
  • Conference Proceedings
  • E. Friedrichs, R. Jungmann, A. Tsokou, S. Renner, F. C. Simmel , Towards in vivo nanomachines, Advances in Science and Technology, 55-58, 120-126 (2008).
  • Jungmann et al. Real-Time Microdamage and Strain Detection during Micromechanical Testing of Single Trabeculae. in Annual Meeting of the Society for Experimental Mechanics. 2007. Springfield, MA: Society for Experimental Mechanics Inc.