OligoTRAFTACs: A generalizable method for transcription factor degradation

Dysregulated transcription factors (TFs) that rewire gene expression circuitry are frequently identified as key players in disease. Although several TFs have been drugged with small molecules, the majority of oncogenic TFs are not currently pharmaceutically tractable due to their paucity of ligandable pockets. The first generation of transcription factor targeting chimeras (TRAFTACs) was developed to target TFs for proteasomal degradation by exploiting their DNA binding ability. In the current study, we have developed the second generation TRAFTACs (“oligoTRAFTACs”) composed of a TF-binding oligonucleotide and an E3 ligase-recruiting ligand. Herein, we demonstrate the development of oligoTRAFTACs to induce the degradation of two oncogenic TFs, c-Myc and brachyury. In addition, we show that brachyury can be successfully degraded by oligoTRAFTACs in chordoma cell lines. Furthermore, zebrafish experiments demonstrate in vivo oligoTRAFTAC activity. Overall, our data demonstrate oligoTRAFTACs as a generalizable platform towards difficult-to-drug TFs and their degradability via the proteasomal pathway.


Cell culture
Human embryonic kidney cells HEK293T cells and HeLa cells were grown in Dulbecco's Modified Eagles Medium (DMEM) containing 10% heat inactivated fetal bovine serum (FBS), streptomycin (5 µg/mL) and 5 U/mL penicillin 95 U/mL). All cell lines were maintained and cell culture experiments were carried out in humidified incubators at 37 degrees and 5% CO 2 supplementation.
Chimeric oligoTRAFTAC transfection was performed using RNAi-Max reagent according

Click rection
Alkyne-modified oligonucleotides were dissolved in ultra-pure water at 500 M concentrations and azide-modified VHL Ligands were dissolved in DMSO at 10 mM. Right before the click reaction, fresh stock solutions of Cu (II) sulfate pentahydrate (50 mM in water), Tris(3-hydroxypropyltriazolylmethyl)amine (THPTA: 100 mM in DMSO) and sodium ascorbate (100 mM in water) were made. The click reaction was carried out in 50% DMSO solution. First, alkyne modified oligonucleotide (250 L) and azide-modified VHL Ligand (1:5 molar ratio) mixed in tube 1. Then, Cu (II) sulfate pentahydrate and THPTA was mixed first, followed by the addition of sodium ascorbate to be final molar ratio of 1:2:2. A 37-fold molar excess of Cu-THPTA complex was added to tube 1 and water and DMSO were added to get the final reaction mixture with 50% DMSO. Click reaction mixture was mixed thoroughly and flushed with inert gas (N 2 ) for 1 minute.
Reaction mixture was then incubated at room temperature for ~ 16 h. Click reaction product was purified by reverse phase high-performance liquid chromatography (HPLC) using a C18 column. HPLC method used for oligo purification (Buffer A-5% acetonitrile,

Western blotting
Protein concentration in all the cell lysates were measured by BCA protein assay kit and equal amounts from each lysate were mixed with 4X loading dye and boiled for 5 minutes followed by 2 minutes centrifugation prior to loading into SDS-PAGE gel. Next proteins on the SDS-PAGE gel were transferred to a PVDF membrane by western blotting and the membrane was blocked with 5% milk in TBST (0.05%Tween 20) for 1 h. Primary antibodies (all Cell Signaling antibodies were diluted 1:1000, c-Myc 1:150) were prepared in TBST with 5% milk and membranes were incubated overnight at 4 0 C. On the following day, membrane was washes for 15 minutes (Incubate for three times, 5 minutes each) and appropriate secondary antibodies (1:5000) were prepared in TBST with 5% milk and incubated with the membrane for 1h at room temperature (RT). Membrane was washed for 30 minutes with TBST (incubate for three times, 10 minutes each) prior to imaging.

EMSA
Click reaction mixtures and unreacted alkyne-modified oligonucleotides were separated in a 1.2 % agarose gel for 1 h at constant 120 mV and DNA bands were captured by the ChemiDoc system (BioRad) using SYBR safe mode. Approximately 1.5 mg of lysate from each sample was incubated with brachyury antibody at 4 o C for 4 h. Protein A agarose beads (30 l) were added to antibody, lysates mixture and rock at 4 o C for ~ 18 h. Beads were washed with 1X TBS for three times with 5-minute incubation during each wash. Immunoprecipitated proteins were eluted by boiling agarose beads in 2X loading buffer (containing 10% ß-ME) for 8 minutes and centrifuged at high speed for 5 minutes prior to the loading into SDS-PAGE gel followed by western blot analysis.

Cell viability assay
Cells were split and subjected to oligoTRAFTAC transfection as described in ''oligoTRAFTAC transfection" method section. Following the transfection, cells were incubated for the appropriate number of days before recording the luminescence reading from the plate reader. CellTiter-Glo reagent was prepared according to the manufactures recommendation and mixed with cell culture medium with 1:1 ratio. CellTiter-Glo reagent (100 L) was added to each well and incubated for 10 minutes before taking the reading.

Zebrafish Husbandry and Microinjection
Tüpfel-longfin zebrafish were raised according to standard protocols approved by the Institutional Animal Care and Use Committee. Experiments were performed before sex determination in zebrafish 1 . Embryos were injected at the one cell stage with 180 picoliters of a 25 µM oligoTRAFTAC solution. They were raised for 48 hours at 28.6 °C and then scored for the presence of tail defects. While injected embryos showed severe, moderate, and mild tail defects, only those with severe and moderate tail defects were considered for quantitation 2 . For western blot analysis, approximately 20 embryos were collected at 8-10 somite stage and deyolked using deyolking buffer (without Calcium: 55 mM NaCl, 1.8 mM KCl, 1.25 mM NaHCO 3 ). Deyolked embryos were centrifuged at 1000 rcf for 30 seconds and the pellet was isolated and washed once with 1 mL of wash buffer (110 mM NaCl, 3.5 mM KCl, 2.7 mM CaCl 2 , 10 mM Tris/Cl pH8.5). After centrifuging at 1000 rcf for 30 seconds, the pellet was lysed in 20 µl of 2X SDS-loading buffer with 10% BME before loading the SDS-PAGE gel.