Biomod/2011/Slovenia/BioNanoWizards/resultstightbindingzfp

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Tight binding ZFPs


Before using purified recombinant chimeric ZFPs to functionalize DNA origami we had to establish their functionality regarding specific binding to their target binding sites on the attachment staples. We used AlphaScreen and electrophoretic mobility shift assay (EMSA) to test the specificity of binding of ZFP chimeric proteins to DNA.


AlphaScreen assay:

AlphaScreen assay is a bead-based proximity assay for study of molecular interactions. Two binding partners of the interaction we wish to establish are bound to separate donor and acceptor beads. Upon photon irradiation donor beads produce singlet oxygen which has 4 microsecond half-life and can diffuse approximately 200 nm into the solution. If the acceptor beads are within that radius, energy is transferred from the singlet oxygen to thioxene derivatives within the acceptor beads, which consequently emit light of a characteristic wavelength. To test zinc finger protein binding to designed DNA attachment hairpins we used Ni2+-NTA-functionalized acceptor beads to bind His-tagged zinc finger chimeras and streptavidin functionalized donor beads to bind either biotinylated staple strands integrated into the DNA origami rectangle or biotinylated hairpins. Biotinylated hairpins are identical to the attachment staples used for integration into the DNA origami rectangle, but are devoid of the standard staple strand sequences. Upon binding of zinc finger proteins to biotinylated DNA origami/hairpins, acceptor and donor beads are brought into the proximity allowing for the singlet oxygen excitation of acceptor beads to take place.

Figure 13: AlphaScreen-based determination of binding of ZFP chimeras to the attachment staples. Our version of Alphascreen assay utilizes Ni2+-NTA donor beads and Streptavidin-acceptor beads. Donor beads bind His-tagged ZFPs, while the biotinylated DNA comprising ZFP attachment sequence binds to streptavidin acceptor beads. Interaction of ZFP with its attachment sequence, brings the donor and acceptor beads together allowing for singlet oxygen to excite acceptor beads which emit the light signal.
Figure 14: AlphaScreen binding assay of GST-2C7, GST-AZPA4, GST-Zif268 and MBP-6F6 to their target DNA sequences. Higher emmited light signals indicate that six-finger ZFPs GST-AZPA4 and GST-2C7 bind to their corresponding DNA sequences with higher affinity than three-finger ZFP GST-Zif268 (equal experimental conditions were used). Non-target biotinylated oligonucleotides were used as negative controls.
The first goal of functional protein characterization with AlphaScreen assay was to confirm binding of zinc finger chimeras to the designed types of DNA hairpins for DNA origami rectangle functionalization. We experimentally confirmed binding of three six-finger (GST-2C7, MBP-6F6 and GST-AZPA4) and one three-finger protein (GST-Zif268) to their target hairpin oligonucleotides, as evident from the Figure 14.

We also checked the specificity of binding ZFPs to their DNA targets. Figure 15 shows the result of a GST-2C7 binding test, where we used four different biotinylated binding site oligonucleotides with binding sequences for Zif268, 2C7, AZPA4 and 6F6. The strongest signal was produced from the experiment of binding of GST-2C7 to 2C7 binding oligonucleotide. A much weaker signal was produced from binding of GST-2C7 to the Zif268 binding site oligonucleotide, which can be explained by the fact that 2C7 binding site is a duplication of Zif268 binding site. No significant interaction was observed with AZPA4 binding site and a signal barely above background was shown with 6F6 binding site.
Figure 15: AlphaScreen cross-reactivity assay of GST-2C7 to other ZFP binding sequences. Result shows that GST-2C7 binds specifically to its designed binding sequence, while it does not bind to oligonucleotides with AZPA4 and 6F6 recognition sequences and only weakly to the Zif268 site, which contains half of its recognition sequence.
Results from AlphaScreen assays therefore confirm that our zinc finger chimeras are soluble, functional and bind to specific DNA sequences and, as such, are suitable for use as DNA origami add-ons.


EMSA

We used electrophoretic mobility shift assay (EMSA) to check the concentration dependence of ZFP binding to their designated DNA targets. Binding of zinc finger chimeras to attachment staples used for DNA origami functionalization was confirmed by observing a shift in oligonucleotide mobility on agarose gel electrophoresis. In the experiment below we checked binding of GST-AZPA4 to type A, B and C attachment staples.

Figure 16: Three types of designed attachment staples for binding ZFP chimeras. DNA duplex segment contains the recognition site for the ZFP protein domain.

The self-annealed binding staples without added protein could be seen at electrophoretic mobility of less than 250 base pairs, according to the ladder, which is consistent with the length and mobility of single stranded attachment staples, i.e. 75, 76 and 80 bases for type A, B and C hairpins, respectively. Increasing the molar excess of protein over attachment staples resulted in complete binding of oligonucleotides. This is apparent from the lack of appropriate bands corresponding to free attachment staples in lanes with higher protein excess added. Mobility shift is apparent from the appearance of a slower migrating band, which was not present in any of the negative controls (attachment staples without protein or protein without attachment staples) and is due to protein binding to attachment staples, which influences the mobility of attachment staples.
Figure 17: EMSA assay demonstrates concentration-dependent binding of ZFP chimeric protein GST-AZPA4 to three types of attachment staples. Separation was performed on 2 % agarose gel and DNA stained with ethidium bromide. The image shows three sets of samples with each set consisting of a binding staple (type A, B or C) with increasing concentrations of GST-AZPA4 (no ZFP, 4x, 8x, 16x molar excess over binding oligonucleotide). The last lane on the right represents a protein sample without added attachment staple. Binding of GST-AZPA4 to attachment staples results in appearance of shifted bands in samples with ZFP (marked with arrows).
 

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