Single-Molecule Imaging to Visualize Fusion Protein Condensates Assembly on DNA Curtains
Transkript
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 DNA-binding green fluorophores. Next, inject a solution of fusion protein tagged to protein-binding magenta fluorophore via its LCD domain, and incubate.
The tagged fusion proteins bind to the target sequence on the lambda DNA to form condensates along the DNA's length. Upon excitation, the DNA-bound and protein-bound fluorophores fluoresce.
The fusion protein condensates appear as magenta-colored puncta on the green fluorescently-labeled double-stranded DNA.
