The AT-Hook motif as a versatile minor groove anchor for promoting DNA binding of transcription factor fragments

We report the development of chimeric DNA binding peptides comprising a DNA binding fragment of natural transcription factors (the basic region of a bZIP protein or a monomeric zinc finger module) and an AT-Hook peptide motif.


Coupling of bromoacetic acid:
The resin containing the peptide (50 mg, aprox. 0.01 mmol) was suspended in dichloromethane (DCM) and shaken for 1 h to ensure a good swelling. On the other hand, bromoacetic acid (28 mg, 20 equiv) was dissolved in 630 µL CH 2 Cl 2 and cooled to 0 ºC. Diisopropylcarbodiimide (DIC, 16 µL, 10 equiv) was added, and the mixture stirred at 0 ºC for 20 min. The solid was filtered off and the flitrate was added over the resin. The suspension was shaken for 30 min. The resin was then filtered and washed with DCM.

Superpositions
This was carried out with MacPymol using the references PDB ID: 1YSA for GCN4, 3UXW for AT-Hook and 1YUI for GAGA.

Fig. S7
Model of the simultaneous interaction of: Left) br (green) and AT-Hook; Right) (SH)GAGA (orange) and AT-Hook.

Oligonucleotide sequence
Double stranded (only one strand is shown) oligonucleotides used for EMSA experiments with conjugates brH and GAGAH were supplied by Thermo Fischer and their sequences were:
TMR-AT•GAGA (TMR-5´-CGCGTCATAATTGAGAGCGC-3´, one strand shown) (5µL, 5 µM) was added to 995 µL of Tris-HCl buffer 20 mM pH 7.5, 100 mM NaCl, 0.02 mM of ZnCl 2 , and the anisotropy was measured. Aliquots of a stock solution in water of brH or GAGAH (12.5 µM) were successively added to this solution, and the anisotropic value was recorded after each addition.
In the case of GAGAH, we also made the experiment in the presence of calf thymus DNA (50 µM in base pairs).

Fig. S8
Plot of the anisotropic values of TMR-AT•GAGA in the presence of calf thymus DNA recorded at 585 nm against the total concentration of GAGAH, and best-fit curve to a 1:1 binding mode with an estimated K D of 42 ± 8 nM. Average of three replicates.
CD measurements were made with a Jasco-715 coupled with a thermostat Nestlab RTE-111. The settings used were: Acquisition range: 300-195nm; band width: 2.0 nm; resolution: 0.2 nm; accumulation: 5 scans; sensitivity 10 mdeg; response time: 0.25 s, speed: 100 nm/min. Measurements were made in a 2 mm cell at 20 ºC. Samples contained 10 mM phosphate buffer pH 7.5 and 100 mM of NaCl, 5 µM peptide and 5 µM of corresponding dsDNA (when present). The mixtures were incubated for 5 min before registering. The CD spectra of the peptide (when measured in the presence of DNA) was calculated as the difference between the spectrum of the peptide/DNA mixture and the measured spectrum of a sample of the DNA oligonucleotide. The spectra are the average of 5 scans and were processed using the "smooth" macro implemented in the program Kaleidagraph (v 3.5 by Synergy Software).

Cellular internalization studies
Cells growing on glass coverslips were incubated in PBS containing 5 µM of TMR-br or TMR-brH for 30 min. Then they were washed twice with PBS and observed in vivo in a fluorescence microscope equipped with adequate filters and differential interference contrast (DIC) microscopy. Digital pictures of the different samples were taken under identical conditions of gain and exposure.
The assay was carried with three different cell lines: monkey Vero cells, A549 human lung carcinoma cells and HeLa cells. .