Biomod/2011/Slovenia/BioNanoWizards

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Our project in 5 minutes

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Nucleic acid nanostructural design is a powerful tool for constructing complex assemblies of different shapes that can be addressed at nanometer resolution. For technological applications DNA origami has to be functionalized in different ways and at selected positions. We significantly extend the repertoire of available modifiers by "DNA origami add-ons", introducing DNA-binding protein domains, which can be selected from hundreds of modular zinc finger proteins (ZFPs). We modified DNA origami by site-specific incorporation of binding staples that contain the recognition site for different ZFPs, thus replacing the need for chemical modifications with self-assembly. An additional advantage of using proteins for DNA origami functionalization is the ability to prepare genetic fusions with variable protein functional domains. Specific binding of ZFP-fusions to DNA origami was demonstrated by AFM. Further step towards applications was provided by the idea and experimental demonstration of vertical DNA origami stacks that can arrange functionalized DNA origami layers in a defined order, which increases the density of integration and represents a technological platform for nanoelectronic components.




Goals of the project

  • Explore the potential of DNA-binding proteins (zinc finger proteins - ZFPs) for the versatile and multiple position-specific addressing on DNA origami.
  • Explore the feasibility of preparing vertical DNA origami stacks using DNA or protein tethers for potential applications.


Achievements

a) We demonstrated proof of the principle for the use of DNA-binding protein domains for adressing specific positions on DNA origami rectangles and introducting selected functional protein domains
b) We designed and experimentally confirmed formation of verticaly stacked DNA origami layers



Those results would not have been possible without solving many smaller challenges:

  • Solve in vitro solubility problem of ZFPs, which is essential for manufacturing protein-derivatized DNA origami. We achieved this by the addition of GST and MBP domains.We constructed, expressed and purified 11 different combinations of short and long ZFPs fused to other protein domains.
  • Prepare functional fusions of ZFPs with yellow fluorescent protein (mCitrine) and Renilla luciferase (RLuc) that can sense the proximity of their DNA-bound positions up to 10 nm distance and generates optical signals without external illumination.
  • Experimentaly determine the selectivity of long zinc finger domain chimeras for their DNA origami attachment staples using AlphaScreen and electrophoretic mobility shift assay (EMSA).
  • Design attachment staples for binding ZFP chimeras to DNA origami.
  • Design DNA biding staples for DNA-tethered vertical stacks.
  • Prepare and purify twin ZFP protein chimeras as DNA origami tethers.


Relevance and applications

Our results provides the foundation for many real world potential applications.The ability of DNA origami to fold into almost any desired shape combined with protein functionality at specific positions represents the perfect marriage for countless applications. Large number of available DNA binding protein domains show promise for simultaneous and specific position-specific addressing of DNA origami.


We expect that ZFP chimeras will push forward the development of high-tech applications such as lab-on-a-nanochip, novel types of artificial biosynthetic organelles, light emitting nanosensors and many others. We extended DNA origami assemblies into the third dimension by formation of vertical stacks where the vertical arrangement is formed based either on DNA or protein tethers. Vertical stacking of DNA origami allows combinations of differently derivatized (e.g. metalized, carbon nanotube derivatized) DNA layers for the formation of various nanoelectronic components, such as nanocapacitors, nanobatteries, ID tags and other devices with dimensions at the nanoscale.

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