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Results of our project experimentally demonstrate the proof of the concept for DNA origami-protein hybrids that could be used in advanced applications

1. Protein add-ons

We enhanced the solubility of zinc fingers so that they can be produced in large amount and used for in vitro applications, rather than only within living cells. Their solubility was increased by genetic fusing them with maltose-binding protein (MBP) or glutathione-S-transferase (GST) domains, which also facilitate purification.

We demonstrated DNA binding of zinc finger domains with increased specificity and affinity based on the recognition of 18 bp binding sequence, which increases their affinity for DNA towards the picomolar range of Kd, making their interaction with DNA almost irreversible. Specific binding to dsDNA attachment staples was demonstrated by AlphaScreen and EMSA experiments.

We demonstrated positional attachment of ZFPs to specific sites on DNA origami.

2. Vertical stacks of DNA origami rectangles

Advanced technological applications of DNA origami may require combinations of more than one type of DNA origami derivatized with different molecules (e.g. conductive carbon nanotubes or metals). For this purpose combinations of two or more DNA origami layers arranged into vertical stacks can be used for fabrication of useful nanoscale devices. Vertical stacking could be accomplished using either DNA or proteins as tethers. The vertical order of DNA origami layers and their number in a stack could be designed at will using different tethers on each side of the DNA origami plates.

We prepared perfectly aligned DNA stack using DNA tethers.

While DNA tethers are relatively easy to implement and ensure their uniqueness, the advantage of protein tethers is that they can provide a fixed distance between stacks due to the discrete size of used protein folds, they are not modified with the same modifiers at the DNA surface and can remain functional.

We designed two different types of protein tethers:

  • twin ZFP chimeras, comprising two different DNA binding domains that ensure anchoring to two different sides or faces of DNA origami and additional solubilizing and purification domains (MBP and His-tag, respectively).
  • heterodimeric ZFPs, comprising of DNA binding domain and a heterodimerizing domain (SH3 peptide and SH3 domain or coiled-coil forming segments) that form a stable heterodimer.

We designed twin ZFP protein tethers, produced them in recombinant form and purified them.

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