Biomod/2011/Slovenia/BioNanoWizards/protdnahybrid

From OpenWetWare

< Biomod | 2011 | Slovenia/BioNanoWizards
Revision as of 17:40, 2 November 2011 by Roman Jerala (Talk | contribs)
(diff) ←Older revision | Current revision (diff) | Newer revision→ (diff)
Jump to: navigation, search

Protein-DNA origami hybrid


Figure 30. AFM image of GST-AZPA4 bound to selected positions on DNA origami rectangles (AFM tapping mode imaging in air). A yellow rectangle marks the origami with all of the three binding sites occupied. Red arrows indicate unbound protein adsorbed to mica.
The central goal of our project was to confirm binding of chimeric zinc finger proteins to attachment staple strands integrated into DNA origami rectangle. We used a DNA origami rectangle design inspired by the original DNA origami paper (Rothemund, 2006, see Methods / DNA origami design). After initial characterization of the correct folding of zinc finger chimeras and characterization of their ability to bind to the designed oligonucleotide hairpins determined by the AlphaScreen and EMSA assay, we proceeded with optimization of conditions for binding to DNA origami rectangle. To achieve successful binding, several factors had to be taken into account:

  • the concentration of protein chimeras added to DNA origami should be in excess, but at the same time low enough to avoid formation of large protein aggregates, which cannot be removed by filtration, since they might interfere with atomic force microscope measurements,
  • excess of attachment staples which are not integrated into DNA origami rectangles had to be removed to minimize the number of potential binding sites for protein chimeras,
  • the time of incubation with DNA origami had to be optimized to allow reaching equilibrium and prevent potential protein inactivation due to air oxidation of cysteine residues of ZFPs (this might be also avoided by incubation in an inert atmosphere).

Successful binding was demonstrated for GST-AZPA4 using the following protocol: DNA origami rectangles were prepared in 5 nM concentration with 5x staple strand excess in 1x TB/Mg2+ buffer. Standard staple strands at positions Bb1, ES3, Be1 were replaced with attachment staples for AZPA4. 1x TB/Mg2+ buffer was removed by filtration and replaced with 35 mM Hepes, pH = 7.0, 50 mM KCl, 12.5 mM MgCl2, 100 uM ZnCl2, 5 mM TCEP and 5 % (v/v) glycerol. DNA origami was incubated with 200x protein excess for 2 h, i.e. 70x excess with respect to the number of available attachment staples per origami, which is also consistent with the results from EMSA and AlphaScreen. Samples for AFM imaging were prepared fresh and imaged in a tapping mode in air. Control was the same DNA origami without added protein.

Figure 31: Specific binding of ZFP chimeras to DNA origami in comparison to control a) DNA origami sample prepared as described. b) DNA origami control without protein.
Results demonstrate that we can bind protein domains to the specific position on DNA origami based on ZFP-DNA interactions. Further experiments are planned to demonstrate simultaneous binding of several different ZFPs to different binding sites on the same DNA origami board.
 

BioNanoWizards - BioMod 2011 team Slovenia. Design by Free CSS Templates.

Personal tools