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AlphaScreen assay

To test the functionality of our chimeric zinc finger proteins we used AlphaScreen™ (PerkinElmer). ALPHA is an acronym, which stands for Amplified Luminescent Proximity Homogeneous Assay. The key components of the assay are two types of coated beads - Donor beads and Acceptor beads. If the two are brought into close proximity, a light signal can be measured. Upon illumination at 680 nm, Donor beads excite ambient oxygen into singlet oxygen via photosensitization. Energy may be transferred from singlet oxygen to thioxene within Acceptor beads if the beads are in close proximity, thus producing a light signal at 520 - 620 nm.

The two types of beads can be brought together by a specific biological reaction. In our tests we used nickel chelate coated Acceptor beads and streptavidin coated Donor beads. Two beads were brought into proximity by a complex of a His-tagged zinc finger protein and biotinylated binding site oligonucleotide. Biotinylated binding site oligos were very similar to origami binding oligos except that they lack the origami staple oligonucleotide segment. They were self-annealed to form a hairpin by keeping a 20 μM solution heated at 95 °C for 10 minutes then leaving it to cool to room temperature. Protein solutions were mixed with self-annealed biotinylated binding site oligonucleotides with final concentrations being 5 μM protein and 167 nM oligonucleotide and final volume 12 μl. In experiments with lower protein concentrations we maintained the same molar ratio of protein and oligonucleotide and the final volume. These mixtures were left at room temperature to allow formation of complexes at a desirable level. After 1 h the mixtures were diluted 50 times in 20 mM Tris pH 7.6, 100 mM NaCl, 5 mM TCEP and 0.1 % BSA. 10 μl of diluted mixtures were transferred into wells of a 384-well plate. A 100-times dilution (from 5 mg/ml to 0.05 mg/ml) of Acceptor beads was prepared in darkness by diluting stock in the same buffer as mentioned before. 5 μl of this dilution were transferred into each well with a sample. The same was done with Donor beads. The plate was covered with aluminum foil and left at room temperature. After 2 hours the light signal was read with PerkinElmer EnSpire™ Alpha 2390 Multilabel Reader.

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