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Protein tethers

In addition to vertical stacking of DNA origami layers using ssDNA we thought of proteins as a tool for achieving the same goal. In particular protein tethering could precisely define the distance between the functionalized DNA origami layers. Many different combinations of distinct twin ZFP chimeras can be rationally designed based on the library of characterized ZFPs (ZiFDB). It is also possible to engineer ZFPs from scratch, especially since the "context-dependent assembly" or CoDA was introduced in 2010 (Sander, 2011), increasing the efficiency of this approach.

As described in the Idea section twin ZFP chimeras could be used to tether DNA origamis into vertical stacks through interactions with modified binding staples extending perpendicularly from the DNA origami. We constructed twin ZFP chimeras in the form of ZFP1 - MBP - ZFP2, where two distinct ZFPs are attached to both termini of the solubility tag MBP.

Figure 35. Protein-based tethering of DNA origami planes with twin ZFP chimeras. (a) Basic element for such protein mediated tethering comprises a solubility enhancing domain (MBP) flanked by two distinct ZFPs. (b) Numerous recombinantly produced twin ZFP chimeras stabilize DNA origami planes at defined distance locking down the stack.

We designed and successfully isolated two such twin-ZFP chimeras, namely 2C7-MBP-6F6 and AZPA4-MBP-6F6. Since MBP acted as a solubility tag we could thus observe high production of soluble protein at the expected size in the cell lysate. However insoluble fraction contained some of the protein, probably due to the presence of two ZFPs domains in the fusion protein. We purified both twin ZFP chimeras, which will be used to prepare protein-tethered DNA origami vertical stacks. The results of the proteins' production, confirmation of their identity by Western blot and purification are presented below.

Figure 36: Production, WB confirmation and isolation of two distinct twin ZFP chimeras. (a) Coomassie Brilliant Blue stain of the soluble and insoluble fractions of the bacterial cell lysate after protein production. (b) Western blot of fractions in the panel (a) using anti-His antibodies. (c) Coomassie Brilliant Blue stain of the twin ZFP proteins purified using chelating chromatography. Grey arrow depicts the position of 2C7-MBP-6F6 (Mr of 86 kDa) and the black arrow the position of AZPA4-MBP-6F6 (Mr of 84 kDa). Proteins were produced under the same conditions as BRET triple fusions: 2x YT medium supplemented with 10 g/L glucose, 100 mg/L antibiotic kanamycin and 0.5 mM ZnCl2 / 30 °C or 37 °C / 160rpm / ~5 or 7 h induction with 1 mM IPTG.

Previous demonstration of the specific binding of GST-AZPA4 protein chimera to a rectangular DNA origami (Tight binding ZFPs, AFM) strongly suggests the isolated twin ZFPs will be able to form hybrid 3D DNA-protein nanostructures.

  • Sander JD, Dahlborg EJ, Goodwin MJ, Cade L, Zhang F, Cifuentes D, Curtin SJ, Blackburn JS, Thibodeau-Beganny S, Qi Y, Pierick CJ, Hoffman E, Maeder ML, Khayter C, Reyon D, Dobbs D, Langenau DM, Stupar RM, Giraldez AJ, Voytas DF, Peterson RT, Yeh JR, Joung JK (2011) Selection-free zinc-finger-nuclease engineering by context-dependent assembly (CoDA). Nature Methods 8: 67-69.

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