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 GFP-BRD4 expression plasmid (Addgene plasmid #65378) was originally obtained from Addgene and kindly provided by a collaborating laboratory.

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