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<body>
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.
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<img
style="font-family: Arial; width: 890px; height: 298px;" alt=""
src="http://openwetware.org/images/d/d8/ProteintetheringFINAL.png"> |
| 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.
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</tbody>
<img
title="http://openwetware.org/images/6/61/TwinZFPsProductionandisolation.png"
style="margin-bottom: 10px; font-family: Arial; width: 890px; height: 328px;" alt=""
src="http://openwetware.org/images/6/61/TwinZFPsProductionandisolation.png"> |
| 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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