Single-Molecule Imaging to Visualize Fusion Protein Condensates Assembly on DNA Curtains

A chimeric oncogenic fusion protein contains the N-terminal low-complexity domain, LCD, of an RNA-binding protein fused with the DNA-binding domain, DBD, of a transcription factor protein. These proteins form a protein condensate, which recruits RNA polymerase-II and drives oncogenesis.

To visualize the assembly of protein condensates by single-molecule imaging, begin with a DNA flow cell ― a fused-silica glass slide fitted with inlet and outlet injection ports. The flow cell's surface is coated with a biotinylated lipid bilayer to protect the surface and fabricated with a zigzag barrier.

Connect it to a microfluidic system. Introduce a solution of streptavidin, which binds to its high-binding affinity ligand, biotin, on the lipid layer. Inject biotin-tagged double-stranded lambda DNA, which contains multiple repeats of fusion protein-binding sequences. The lipid bilayer's streptavidin binds to the DNA's biotin and attaches the lambda DNA to the bilayer.

Flush with a suitable buffer. Its flow's hydrodynamic force straightens the anchored DNA molecules, allowing them to hang as DNA curtains over the barriers.

Place the flow cell under a fluorescence microscope. Inject a solution of