brd4 bromodomain plasmid  (Addgene inc)

Journal: bioRxiv

Article Title: Microfluidic Agarose µ -Droplets for DNA-Encoded Chemical Library Screening

doi: 10.64898/2026.02.15.706034

Figure Lengend Snippet: (A) Chemical structure of the JQ1-oligo-Cy5 conjugate used as a fluorescent probe (BP). JQ1, a selective BRD4 ligand, is covalently linked to a DNA oligonucleotide and a Cy5 fluorophore, allowing fluorescence-based detection of BRD4 binding. (B) Schematic illustrations (top) and corresponding fluorescence images (bottom) of agarose μ-droplets containing magnetic beads only, BRD4-bound magnetic beads, agarose alone, intact HeLa cells, or permeabilized HeLa cells after incubation with JQ1-oligo-Cy5. Strong Cy5 fluorescence was observed in droplets containing BRD4-bound magnetic beads and permeabilized HeLa cells, whereas no detectable Cy5 fluorescence was observed in magnetic beads only, intact HeLa cells, and agarose-only droplets. Scale bars, 50μm. (C) Schematic illustration of two-color Exchange-PAINT imaging using orthogonal DNA docking-imager strand pairs. An R6 docking strand conjugated to an anti-GFP nanobody was used to label GFP-BRD4, while an R2 docking strand was incorporated into the JQ1-based probe (JQ1-BP). Sequential super-resolution imaging was performed using R6* and R2* imager strands to independently localize BRD4 and bound JQ1, respectively. (D) Two-color Exchange-PAINT super-resolution images of GFP-BRD4-expressing Cos7 cells showing GFP-BRD4 (green) and the JQ1-based probe (JQ1-BP, red). Left, whole-nucleus view; middle, magnified view of the boxed region highlighting nanoscale clustering of BRD4 and JQ1. Right, the same region after Q-PAINT-based cluster filtering (K > 10), revealing higher-order nanoclusters containing both BRD4 and JQ1. Enrichment of JQ1-BP localizations at GFP-BRD4 clusters indicates that JQ1 preferentially localizes to BRD4-enriched nuclear regions.

Article Snippet: The BRD4 bromodomain plasmid (pHis-BRD4 BD1, item #196544), GFP-BRD4 expression plasmid (GFP-BRD4, item #65378) was obtained from Addgene (Watertown, MA, USA).

Techniques: Fluorescence, Binding Assay, Magnetic Beads, Incubation, Imaging, Expressing

Journal: bioRxiv

Article Title: Microfluidic Agarose µ -Droplets for DNA-Encoded Chemical Library Screening

doi: 10.64898/2026.02.15.706034

Figure Lengend Snippet: (A) Composition of the four-compound library used for proof-of-concept DEL screening, including JQ1 as a positive control, GL-CBS and methotrexate (MTX) as off-target binders, and benzoic acid as a negative control. (B) Enrichment ratios (Target / Control) derived from nanopore sequencing data following small-scale DEL screening in agarose μ-droplets containing BRD4-bound magnetic beads (target) or magnetic beads only (control). JQ1 exhibited strong enrichment above the threshold (Target / Control = 1), whereas other compounds showed minimal enrichment. (C) Quantitative analysis of small-scale DEL screening performed in cell-containing agarose μ-droplets, in which DEL enrichment was quantified by qPCR following screening. Agarose-only μ-droplets were used as a control condition, while μ-droplets containing permeabilized HeLa cells or BRD4-overexpressing HeLa cells were treated as target conditions. JQ1 showed the highest enrichment among tested compounds and displayed a pronounced preference for BRD4-overexpressing HeLa cells compared to permeabilized cells. Representative bright field, emGFP, and Cy5 fluorescence images are shown on the right. Scale bars, 50μm. emGFP fluorescence was observed specifically in BRD4-overexpressing HeLa cells. As both HeLa cell conditions were permeabilized, Cy5 fluorescence originating from BP was detected in droplets containing either permeabilized or BRD4-overexpressing HeLa cells.

Article Snippet: The BRD4 bromodomain plasmid (pHis-BRD4 BD1, item #196544), GFP-BRD4 expression plasmid (GFP-BRD4, item #65378) was obtained from Addgene (Watertown, MA, USA).

Techniques: Drug discovery, Positive Control, Negative Control, Control, Derivative Assay, Nanopore Sequencing, Magnetic Beads, Fluorescence

Journal: bioRxiv

Article Title: Microfluidic Agarose µ -Droplets for DNA-Encoded Chemical Library Screening

doi: 10.64898/2026.02.15.706034

Figure Lengend Snippet: (A) Aggregated nanopore sequencing read count distributions for each core scaffold across four screening conditions: magnetic bead–only droplets, BRD4-bound magnetic bead droplets, permeabilized HeLa cell droplets, and BRD4-overexpressing HeLa cell droplets. The DNA-encoded library consisted of three distinct core scaffolds (pyrimidine, trifunctional benzene, and chiral proline cores), each comprising a 96 × 96 × 96 combinatorial spaces. (B) Three-dimensional enrichment maps for each core scaffold across the four screening conditions, displaying the top 200 BB1–BB2–BB3 combinations ranked by nanopore sequencing read count. Each axis (x, y, and z) corresponds to one building block position (BB1, BB2, and BB3), and individual points represent distinct BB1– BB2–BB3 combinations. Point color indicates read count abundance, while combinations exceeding a read count threshold of 65 are additionally highlighted by increased marker size to emphasize highly enriched species. (C) Chemical structures of the top enriched compounds identified from large-scale DEL screening. Enrichment was calculated as the ratio of nanopore sequencing read counts between target and control samples. For each core scaffold, the two compounds with the highest target-to-control enrichment are shown for both magnetic bead droplet and HeLa cell droplet screening conditions. The corresponding BB1-BB2-BB3-derived structures are displayed.

Article Snippet: The BRD4 bromodomain plasmid (pHis-BRD4 BD1, item #196544), GFP-BRD4 expression plasmid (GFP-BRD4, item #65378) was obtained from Addgene (Watertown, MA, USA).

Techniques: Nanopore Sequencing, Blocking Assay, Marker, Control, Derivative Assay