DOCUMENTOS Y REGISTROS DEL SIPLAFT

Several photo-activated zinc finger nucleases have been reported, the most common being photo caged zinc finger nucleases103 _{using, for example, an ortho-nitrobenzyl}

group as a bulky protecting group to prevent enzyme activity.21 _{When subsequently}

removed with UV irradiation, the completely inert protein was converted into one with 70% of the activity of the wild type species.4 _{Unfortunately, irradiation of ortho-}

nitrobenzyl groups releases nitrate by-products which can be toxic to some cells.10

Combined with the harmful UV light these by-products significantly reduce cell viability.

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Figure 54. Photo-decaging of a tyrosine residue.

This process only allows for the selective activation, not deactivation of the nuclease; a drawback common to all decaging procedures. As a single -helix is primarily responsible for ZF-TF binding to DNA it was chosen for modification to allow bidirectional control using an azobenzene photoswitch using an i,i+7 cystiene spacing for low background activity.

2

2..44..11 DDeessiiggnn ooff tthhee SSlluugg ffiinnggeerr ppeeppttiiddeess

The initial aim of the project was to take the zinc finger transcription factor and excise only the residues from the -helix that bind in the major groove of DNA. The wild type Slug zinc finger protein contains four canonical Cys2His2 zinc fingers plus a fifth

atypical zinc finger (Figure 55).

MPRSFLVKKH FNASKKPNYS ELDTHTVIIS PYLCESYPMP VIPKPEILTS GAYSPITVWT SAVPFHSPLP SGLSPLTGYS SSLGRVSPLP SSDTSSKDHS GSESPISDEE ERLQPKLSDP HAIEAEKFQC NLCNKTYSTF SGLAKHKQLH

CDAQARKSFS CKYCDKEYVS LGALKMHIRT HTLPCVCKIC GKAFSRPWLL

QGHIRTHTGE KPFSCPHCNR AFADRSNLRA HLQTHSDVKK YQCKNCSKTF SRMSLLHKHE ESGCCVAH

Figure 55. Sequence of the product of the SNAI2 gene in Homo sapiens (UniProtKB: O43623). The chosen DNA-binding helices are highlighted in red and blue.

Zinc finger peptides

The Slug sequence was homology modelled onto the archetypal zinc finger ZIF268 (PDB 1AAY) using SwissModel online tools and the fingers that aligned most closely to the ZIF 268 sequence were selected for initial testing (Figure 56).

Figure 56. Cartoon of SNAI2 zinc finger residues after homology modelling their sequence onto the structure of ZIF268 (PDB 1AAY). Full schematic (left) -helical regions only (right).

The sheets and loops were removed as the photoswitch will fulfill the role of zinc ion in adding rigidity to stabilise the helix. This leaves only the short -helix regions that make the primary contacts with DNA bases during binding. Lysine and histidine residues in each helix were replaced by cysteines to create an i,i+7 spaced pair for the incorporation of a photoswitch (Table 6). These residues were chosen because they face away from the DNA binding faces and should not significantly affect the binding of the peptide to DNA. An additional peptide was designed with these two helices joined by a short linker (Figure 57).

Figure 57 Double slug with photoswitch.

Sequence Ac-AWLQGHIRTHTG-NH

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Double Slug Finger Ac-AWCQGHIRTCTGEGGDRSNCRAHLQTCSD-NH2

Table 6. Sequences of Slug zinc fingers (Linker in red, original residues in blue, modified in green).

The short glycine-based linker was chosen because of its flexibility to allow the - helices to align with the major grove of the DNA. They were called Slug Finger 1, Slug Finger 2 and Slug Double Finger (Table 6).

2

2..44..22 SSoolliidd ssttaattee ppeeppttiiddee ssyynntthheessiiss

Figure 58. Elementary steps of solid phase peptide synthesis.

Peptides were assembled iteratively by solid phase peptide synthesis using standard 9- fluorenylmethyloxycarbonyl (Fmoc) protected amino acid building blocks with orthogonal acid-labile sidechain protecting groups. This method of forming a peptide bond is approximately 95-99 % efficient, with diminishing returns limiting its practical application to between 20 and 60 amino acids.

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Figure 59. Cleaving of peptide from a solid support with the use of TFA (where R is the peptide).

Once chain extension was complete, the N-terminus was deprotected with piperidine, then the resin washed and amine protected as an acetamide using acetic anhydride. The peptide was then cleaved from the resin and the amino acid sidechains simultaneously deprotected with trifluoroacetic acid containing tri-iso-propylsilane (5%), water (5%) and 3,6-dioxa-1,8-octanedithiol (5%) scavengers (Figure 59). After half an hour, the reaction mixture was filtered to remove the spent resin and the TFA was removed under a stream of nitrogen and the residue dispersed in diethyl ether and chilled in a freezer overnight. Filtration provided a crude peptide mixture that was purified by HPLC as described in the methods section. Purified peptides were then checked by matrix-assisted laser desorption ionisation/time of flight (MALDI-TOF) mass spectroscopy to ensure they gave masses corresponding to the intended products (Table 7).

Table 7. Expected and observed masses for Slug peptides.

Calculated

mass Observed mass

Slug Finger 1 1373.60 1373.44a

Slug Finger 2 1531.72 1533.89a

Double Slug Finger 3204.56 3185.85 [M –H2O]

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2..44..33 SSyynntthheessiiss ooff ssuullffoonnaatteedd aazzoobbeennzzeennee ccrroosssslliinnkkeerr

Figure 60. 3'-Bis(sulfonato)-4,4'-bis(chloroacetamido)azobenzene (71) was chosen for its water solubility.

Figure 61. Scheme showing the synthesis of BSBCA (71).104

The first step is a selective acetate protection with acetic anhydride under acidic conditions. Selectivity is achieved because the sulfonate group changes the pKa of the

amine in the ortho position, making it harder to protonate. In initial experiments performing the reaction below 90 o_{C resulted in a 50:50 mix of both the singly and}

doubly acetylated protected species, but rigorously maintaining the temperature at 94 C greatly improved the ratio for the desired monoacetate. Oxidative coupling then converted two molecules of an aniline containing molocule (91) to azobenzene (Figure 62).

Zinc finger peptides

Figure 62. Mechanism for oxidative coupling of anilines to form azobenzene.

The reaction proceeds through oxidation of the amine by hypochlorite to a hydroxylamine, then to a nitroso group followed by condensation with another molecule of amine. The acetate protecting groups were then removed using hydrochloric acid and the resulting solution was neutralised and lyophilised. The free amine was then converted into the chloroacetamide by reaction with chloroacetic anhydride to furnish 71 in an overall yield of 1.3 %. The UV/visible absorbance spectra of 71 displayed distinct extinction coefficients for the -* transitions of the trans and cis isomers (Figure 63).

0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 300 350 400 450 500 550 600 Ab so rb an ce Wavelength / nm

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