DNA cleavage studies with engineered Zf13 domains

The aforementioned DNA-binding experiments for the synthesized zinc finger mutants 29 and 30 were promising with regard to a possible phosphodiester hydrolysis. An already proceeded DNA cleavage during the previously described experiment could not be excluded due to the use of fully operational peptides. However, the applied native reaction conditions

prevented the detection of such an event. As mentioned at the beginning, the implementation of the building blocks at only one distinct position in the peptide just enabled the possibility for the latter to reach the phosphate groups of exactly one DNA strand. For this reason, a double-strand cleavage was virtually impossible. Hence, a single-strand cleavage could not be detected due to still existing WATSON-CRICK base pairing holding the duplex together over the full length of the DNA 30-mer. In addition, the incubation time of 2 h, which was applied in the previous binding studies, was rather short to scissor major amounts of the DNA substrate. This assumption was confirmed by the results obtained from the hydrolysis studies with the activated, and therefore, easier to cleave BNPP model substrate. Consequently, the PAGE conditions had to be adjusted to non-native conditions in order to visualize the formation of cleavage products.^[97] The peptides were prepared according to the method described in section 2.6. After protein dialysis, incubation was carried out in a sample buffer (20 mM Tris, 150 mM NaCl, 0.5 mM TCEP, 1 M ZnCl2, pH 7.8) including the zinc finger mutants and duplex DNA (0.5 M) under native conditions and for 72 h instead of 2 h.

Moreover, the temperature was increased from room temperature to 37 °C in order to further increase hydrolysis rates. Subsequently, denaturing conditions were applied by preparing a 1:1 dilution of the sample probes with a double-concentrated solution containing SDS (2%), glycerol (25%, v/v) EDTA (2 mM) and DTT (180 mM) in Tris buffer (20 mM Tris, 150 mM NaCl, 0.5 mM TCEP, pH 7.8). The mixtures were heated to 95 °C for 5 min and were subsequently loaded on the gel after cooling on ice. Under these conditions, the peptides as well as the duplex DNA were completely denatured that goes in hand with the release of smaller DNA fragments resulting from single-strand cleavages. Different peptide to DNA ratios (rf = CZf13/CDNA = 20, 30 and 60) were examined, and therefore, prepared in parallel. After loading of the samples on the gels, electrophoresis was performed using a denaturing running buffer (25 mM Tris, 192 mM glycine, 0.1% SDS, pH 8.2).

The identification of the most beneficial incorporation sites for the building blocks was based on molecular models, which were generated using published crystallographic data. As mentioned in section 2.4, the building blocks incorporated at position 70 should cleave the operator strand, whereas the building blocks at position 75 should cleave the opposite strand of the DNA. As a consequence, fluorescent labeling of just the individual single-strand, which takes part in the hydrolysis reaction, appears appropriate but bears the risk of causing a false negative result in case the other strand had been cleaved instead. In order to avoid misleading interpretations, all experiments were performed twice with either the binding-strand labeled with 5'-FAM (Figure 2.17) or with the opposite non-binding binding-strand (Figure 2.18) labeled with the same fluorescent dye.

The control lane contains solely the duplex DNA without any addition of unmodified zinc finger due to its documented lack of hydrolysis capacity and because of peptide wastage by virtue of the denaturing conditions. Hence, the only band observed after electrophoresis displayed the fully intact and labeled DNA single strand.

The results obtained for the cleavage experiment with the labeled operator strand and the zinc finger mutants 29 and 30 incorporated at position 70 showed only bands on the same level with respect to the control lane. Moreover, also the peptides 31 and 32, which were incorporated at position 75, did not show any significant differences in terms of their migration speed indicating the integrity of the labeled DNA single strand. Also, the experiments that labeled the opposite DNA strand, revealed the same results (Figure 2.18).

Consequently, all experiments performed with the four zinc finger mutants showed an unverifiable hydrolysis ability under the applied reaction conditions. This was further

rf 20 30 60 20 30 60

# 1 3 4 5 7 8 9

rf 20 30 60 20 30 60 ref. Zf13BMIA70 Zf13BPA70 ref. Zf13BMIA75 Zf13BPA75

# 1 3 4 5 7 8 9

Figure 2.17 Denaturing PAGE studies to examine the cleavage ability of the engineered zinc fingers towards their dsDNA target-site. Different peptide to DNA ratios (rf = CZf13/CDNA = 20, 30 and 60) were prepared and incubated at 37 ° for 72 h. The single-stranded DNA oligomer containing the operator sequence for the zinc finger was 5′-labeled with a FAM fluorophore in order to visualize fragmented DNA.

supported by control experiments in which the incubation time was prolonged to 5 days and the temperature was increased to 55 °C.

Finally, the question why the examined zinc finger mutants were not able to perform hydrolysis on the consensus DNA sequence, even though their binding abilities had been proven for DNA model substrate, is to be clarify.

One aspect may be a possible insufficient substrate binding, and therefore, an inadequate activation of the latter. The phosphodiester backbone of the DNA is particularly stable even at high concentrations of hydroxide ions.^[98] Without an appropriate activation by a dinuclear metal complex, which forces hydrolysis in the direction of phosphodiester cleavage, the cleavage mechanism generally is a reversible process. It is even conceivable that due to the relative rigidity of the peptide structure bound to DNA, the building blocks are not perfectly able to bind the substrate. X-ray crystallography of the present DNA/peptide complex could provide useful information on the behavior of the building blocks when bound to DNA. This would allow for a more profound interpretation of the actual factors leading to an absence of DNA hydrolysis.