DNA cleavage studies with phosphoserine modified Zf13 peptides and Ce(IV)/EDTA

In a similar approach, the site-specific hydrolysis ability of the Ce(IV)/EDTA complex towards double-stranded DNA was under evaluation using denaturing PAGE.^[97] In general, the latter relies on the increased accumulation of the hydrolytically active metal complex at the phosphoserine species, which occupies a distinct position in the peptide. Thus, upon binding of the zinc finger mutants to the dsDNA, the phosphate side-chain of phosphoserine comes in close proximity to the phosphodiester backbone of the DNA. This situation promotes the relocation of the phosphoserine bound Ce(IV)/EDTA complex to the neighboring phosphodiester backbone. Consequently, DNA fragments of predictable molecular size should originate from the cleavage that have a different migration speed in the PAGE assay in comparison to the intact DNA single-strands.^[142]

Prior to the experiments, peptides 43 and 44 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 the consensus duplex DNA at room temperature for 3 h. Based on the findings obtained from the previous DNA-binding studies (section 3.5), the most suitable peptide to DNA ration (rf = 20) was applied and kept constant in order to examine the influence of different Ce(IV)/EDTA concentrations on the DNA cleavage. Hence, a buffered stock-solution of Ce(IV)/EDTA (20 mM, 20 mM Tris, 150 mM NaCl, 0.5 mM TCEP, pH 7.8) was added to the Zf13/DNA samples to obtain the metal complex in final concentrations of [M] = 10, 20, 50, 100 or 200 ^M.The probes were incubated under the exclusion of molecular oxygen at 37 °C for 72 h before denaturing conditions were applied by preparing a 1:1 dilution of the samples 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 immediately loaded on the gel after cooling on ice. Electrophoresis was performed using a denaturing running buffer (25 mM Tris, 192 mM glycine, 0.1% SDS, pH 8.2) and the migrated DNA was visualized by a fluorescence imager, which detected the attached 5'-FAM label. The latter was either attached to the binding strand (Figure 3.16), which included the operator sequence for the zinc finger peptides, or to the opposite strand (Figure 3.17). Consequently, the aforementioned procedure was performed in parallel for the two differently labeled dsDNA samples under the same condition.

Figure 3.16 shows the findings for the experiment with dsDNA having the label attached to the binding strand. Well 1 in each gel represents the reference, which contained only the dsDNA without further addition of peptide or Ce(IV)/EDTA. Wells 3, 4, 5, 6 and 7 contained the engineered zinc fingers, either with the phosphoserine residue incorporated at position 70 or 75 as well as with different Ce(IV)/EDTA concentrations ([M] = 10, 20, 50, 100 and 200 M). It is conspicuous that all migrating bands were on comparable levels, which also included the reference. This strongly accounts for consistently intact single-stranded DNA samples that were not prone to hydrolysis over the contemplated reaction time. This result was also observed for the similar experiment including the dsDNA bearing the fluorescent label at the opposite strand (Figure 3.17). The experiments were repeated with a prolonged incubation time of 120 h instead 72 h but with the same experimental result. Also a mass spectrometric examination of the samples after the incubation showed predominantly the intact oligomers.

[M] conc. 10 20 50 100 200

# 1 3 4 5 6 7

[M] conc. 10 20 50 100 200

# 1 3 4 5 6 7

ref. Zf13Pser70 ref. Zf13Pser75

Figure 3.16 Denaturing PAGE studies to examine the cleavage ability of the phosphoserine modified zinc fingers towards their dsDNA (0.5 M) target-site. The peptide to DNA ratio was fixed to rf = CZf13/CDNA = 20 for all samples. The Ce(IV)/EDTA concentration was varied ([M] = 10, 20, 50, 100 or 200 M). Incubation was performed at 37 °C for 72 h. The single-stranded DNA oligomer containing the operator sequence was 5′-labeled with a FAM fluorophore in order to visualize fragmented DNA.

The limited cleavage ability for the zinc finger bearing the phosphoserine at position 75 can be at least partially attributed to its distorted secondary structure. The latter was presumably responsible for the limited DNA-binding ability as confirmed by native PAGE. For this reason, it is not surprising that Zf13Pser75 had no tendency to cleave DNA because the zinc finger was not able to recruit the catalytically active species to the desired target site. The absence of cleavage products in the PAGE studies observed for Zf13Pser70 with the phosphoserine residue at position 70 is not so general to answer. Possible reasons are summarized in section 3.7 and a discussion is subject to section 4.

[M] conc. 10 20 50 100 200 [M] conc. 10 20 50 100 200

# 1 3 4 5 6 7

ref. Zf13Pser70

# 1 3 4 5 6 7

ref. Zf13Pser75

Figure 3.17 Denaturing PAGE studies to examine the cleavage ability of the phosphoserine modified zinc fingers towards their dsDNA target-site (0.5 M). The peptide to DNA ratio was fixed to rf = CZf13/CDNA = 20 for all samples. The Ce(IV)/EDTA concentration was varied ([M] = 10, 20, 50, 100 or 200 M). Incubation was performed at 37 °C for 72 h. The single-stranded DNA oligomer opposite to the operator-sequence-containing strand was 5′-labeled with a FAM fluorophore in order to visualize fragmented DNA.

3.7 Summary

This section focused on the concept of modifying the DNA-binding zinc finger domain by the incorporation of a phosphoserine residue in order to recruit external Ce(IV)/EDTA complexes to a desired target site. The documented high hydrolysis capacity of lanthanide complexes should be thus utilized to trigger DNA cleavage.^[104] The lack of reliable values that describe the molecular attraction of the phosphoserine moiety towards the Ce(IV)/EDTA complex was successfully overcome by the establishment of a microscale thermophoresis approach. In doing so, the phosphoserine residue was coupled to a PEG spacer, which was equipped with a FITC fluorophore. A similar system with unmodified serine served as a reference sample and both moieties were used to determine the dissociation constants for the corresponding complexes with Ce(IV)/EDTA by means of fluorescence quenching. It was clearly demonstrated that only the phosphorylated serine residue was able to form a complex with the hydrolytically active species, whereby it was possible to calculate a profound dissociation constant. This simplified system served as benchmark for the evaluation of the phosphoserine modified zinc finger 3 domain. It was found that the dissociation constant obtained for the Zf3/[Ce(IV)/EDTA] complex was in a comparable range implying an increased accumulation of the lanthanide complex in close proximity of the phosphoserine residue. These findings were beneficial in conjunction with the binding of the zinc finger to its consensus DNA sequence, which predetermines possible hydrolysis sites with regard to the location of the phosphoserine in the peptide sequence. Thus, CD spectroscopic studies were performed in order to examine the influence of the unnatural amino acid on the secondary structure formation of the zinc finger peptide. As previously observed for the incorporation of the internal dinuclear building blocks (section 2.4.4), the incorporation at the arginine 70 position provided best results with respect to the formation of the required -structure. In contrast, the incorporation at the serine 75 position resulted in a deviated CD spectrum, which indicated a negative influence of the phosphoserine on the peptide folding.

Moreover, the phosphoserine modified Zf3 mutants were successfully used in an EPL approach to generate the full-length three tandem zinc-finger domain of Zif268 with enhanced DNA binding ability. With regard to the native zinc finger reference sample, PAGE studies under non-denaturing conditions successfully revealed a similar peptide/DNA complex formation ability for the engineered zinc finger with the phosphoserine residue incorporated at position 70. The disturbed peptide folding ability, which was found for the mutant modified at position 75 was further proven due to its apparently poor ability to interact with DNA.

A similar experiment should shed light on the hydrolysis activity of the engineered zinc finger peptides or to be more precise, on the site-specific cleavage by the recruited Ce(IV)/EDTA

complex. Hence, the dsDNA samples were incubated with the ZF-mutants and different amounts of the hydrolytically active Ce(IV)/EDTA complex were added. After the incubation at 37 °C and for different reaction times varying from 72 – 120 h, there was no indication for phosphodiester hydrolysis observed after gel electrophoresis. A possible explanation might be a limited substrate accessibility of the recruited lanthanide species. The preformed zinc-finger/DNA complex could be to compact to additionally allow the binding of the bulky Ce(IV)/EDTA complex to the partially shielded phosphoserine residue. During the MST experiments, solely the individual zinc fingers were examined for their ability to bind the metal complex. The additional use of DNA was neglected due to the possible occurrence of a peptide/DNA-derived binding event, which might superimpose the actual target. The changed conditions after DNA binding might have caused a lowered accessibility of the phosphoserine residue leading to an unspecific movement or binding of the active species. Consequently, the utilization of its full hydrolytic potential, even at elevated concentrations, was inhibited.

The actual conditions after the peptide/DNA complex formation need to be evaluated by means of X-ray crystallography in order to develop a detailed understanding. Due to the fact that the hydrolysis capacity of the examined cerium complex was multiply demonstrated in literature, this gives reason to review the structural conditions, such as the incorporation site of the phosphoserine residue in the zinc finger in order to establish a functional system.

However, the results obtained from this approach can help to refine the current system to develop a powerful artificial restriction endonuclease with enhanced site-specificity.