This study investigates the assembly of protein condensates formed by a chimeric oncogenic fusion protein. The research employs single-molecule imaging techniques to visualize the interaction of these proteins with DNA.
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.
To image the EWS-FLI1 condensation formation on DNA curtains, open the imaging software and find and mark the positions of the nine zig-zag patterns under bright-field. Then, turn on the flow at 0.2 milliliters per minute to stain the DNA with double-stranded DNA dye for 10 minutes.
Next, dilute the mCherry-EWS-FLI1 protein with the imaging buffer at a concentration of 100 nanomoles and 100 microliters. Then, load the protein sample through the valve with a 100-microliter glass syringe, and change the flow rate to 0.4 milliliters per minute.
After turning on the 488-nanometer laser, pre-scan each region to check the DNA distribution state, and select the region in which the DNA molecules distribute evenly. Then, set the laser power to 10% for the 488-nanometer laser and 20% for the 561-nanometer laser, and using the power meter, measure the real laser power near the prism.
Start acquiring images at two-second intervals with both 488 and 561-nanometer lasers simultaneously. Then, change the valve from the manual mode to injection mode to let the imaging buffer flush the protein sample into the flow cell after 60 seconds.
To remove the free EWS-FLI1, keep washing the flow cell with the imaging buffer for five minutes with only the 561-nanometer laser switched on. Then, stop the flow and incubate at 37 degrees Celsius for 10 minutes.
After 10 minutes, turn on the flow at 0.4 milliliters per minute to let the DNA extend, and acquire images at two-second intervals between different frames to obtain high-throughput data of EWS-FLI1 condensate formation.
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