biotin tagged dna probes  (Sangon Biotech)

Journal: iScience

Article Title: Development of small molecule inhibitors targeting PBX1 transcription signaling as a novel cancer therapeutic strategy

doi: 10.1016/j.isci.2021.103297

Figure Lengend Snippet: PBX1 inhibitors destabilize the PBX1-DNA binding complex (A) Docking analysis of the PBX1 inhibitor of the PBX1 protein-DNA contact interface. Left: An overview of the DNA-PBX1 interaction site and T417 docking pose. Middle: A zoomed-in view of the rectangular box in the overview figure shows the distances between the T417 side chain (pink-red) and specific amino acid residues of the PBX1 protein (green-blue). Right: Docking analysis of T417 on the contact interface of PBX1 protein. T417 contacts H205 (blue), K266 (yellow), R288 (red), I289 (green), and K292 (cyan) of the PBX1 protein. (B) Small molecule inhibitors were designed based on docking to the crystal structure of the PBX1-DNA contacting interface. The potency of the newly designed small molecules in destabilizing the PBX1-DNA complex was assessed by EMSA. Candidate inhibitors were serially diluted and incubated with purified PBX1 protein and biotin-labeled oligonucleotides containing consensus PBX1 binding motif sequences. The half maximal inhibition concentration (IC 50 ) of the lead compounds, T417, T418, and T383, was determined by quantification of band intensities.

Article Snippet: Biotin-tagged double-stranded DNA probes, the same probes used for EMSA assays, were immobilized on a Biacore sensor chip that was coated with neutravidin.

Techniques: Binding Assay, Incubation, Purification, Labeling, Inhibition, Concentration Assay

Journal: iScience

Article Title: Development of small molecule inhibitors targeting PBX1 transcription signaling as a novel cancer therapeutic strategy

doi: 10.1016/j.isci.2021.103297

Figure Lengend Snippet: Cellular thermal shift assay (CETSA) and surface plasmon resonance (SPR) assay for evaluation of drug-PBX1 protein interaction (A) Western blot analysis of cellular thermal shift assays (CETSAs). SKOV3 cells stably expressing the PBX1-V5 protein were treated with 50 μM of each compound for 1 h and subsequently heated at 37°C, 40°C, 43°C, 46°C, 49°C, 52°C, 55°C, or 58°C for 3 min. Soluble PBX1 protein bound to the test compound was visualized by western blot using an anti-V5 antibody. The detailed protocol is provided in . (B) Quantification of CETSA melt curves based on the PBX1-V5 band intensities shown in (A). Data are normalized to band intensities obtained at 37°C. (C) Bar graph showing the results of CETSAs measured at 52°C and 58°C. Data represent means ± SD. (D) SPR sensogram showing purified PBX1 protein (PBX1-DBD) binding to immobilized double-stranded DNA probes containing PBX1 binding motif (left) or without PBX1 binding motif (right) at six concentrations, ranging from 3.125 to 100 μM in triplicate. (E) Test efficacy of small molecular inhibitor T417 or its analog in interfering with the PBX1 protein-DNA probes interaction. SPR sensogram shows PBX1 protein (PBX1-DBD) (red), PBX1 protein + inhibitor (blue), and inhibitor (green) binding to the immobilized DNA probes containing the PBX1 binding motif. Each experiment was performed in duplicate. (F) T417 competition of PBX1 protein binding to immobilized PBX1 consensus DNA at nine concentrations ranging from 0 to 80 μM in duplicate. Data are normalized to binding in the absence of T417 inhibitor. The IC 50 derived is 5 μM.

Article Snippet: Biotin-tagged double-stranded DNA probes, the same probes used for EMSA assays, were immobilized on a Biacore sensor chip that was coated with neutravidin.

Techniques: Thermal Shift Assay, SPR Assay, Western Blot, Stable Transfection, Expressing, Purification, Binding Assay, Protein Binding, Derivative Assay

Journal: iScience

Article Title: Development of small molecule inhibitors targeting PBX1 transcription signaling as a novel cancer therapeutic strategy

doi: 10.1016/j.isci.2021.103297

Figure Lengend Snippet: T417 selectively suppresses the PBX1/MEIS2 transcriptional complex (A) The DNA probes were generated to recognize either the PBX1 binding motif only, TTGAT, or to recognize the PBX-MEIS heterodimeric motif, TTGGATTGACAG. Mutant probes of PBX or MEIS were also generated as negative controls. (B) Verification of probes. Purified PBX1 and MEIS2 proteins were incubated with different probes, and formation of a trimeric complex of PBX1/MEIS2/DNA was assessed by EMSA. Complexes were not detected when mutant probes were employed. (C) Supershift assay to confirm binding specificity. We added 0.5 or 1 μg antibodies against PBX1 or MEIS2 to the reaction. (D) Binding inhibition of the PBX1/MEIS2/DNA complex by T417. Serial dilution of T417 was incubated with the complex and analyzed by EMSA ( left ). Quantification of EMSA image band intensity ( right ).

Article Snippet: Biotin-tagged double-stranded DNA probes, the same probes used for EMSA assays, were immobilized on a Biacore sensor chip that was coated with neutravidin.

Techniques: Generated, Binding Assay, Mutagenesis, Purification, Incubation, Inhibition, Serial Dilution