Single-Molecule Imaging of EWS-FLI1 Condensates Assembling on DNA

We provide a single molecule assay to observe the phase separation of transcription factors on DNA This technique allows realtime realization of the dynamic process of transcription factors assembling as a liquid droplet on DNA. EWS-FL1 is one of the most frequently involved fusion genes in Ewing sarcoma tumorigenesis. Our research could identify the relationship between DNA motif number and EWS-FL1 condensate formation which is associated with a barren gene transcription in tumor cells, and maybe helpful for the prognosis.

This method could be applied to study any transcription condensates or complexes in vitro, like P53 and MED1. To prepare a flow cell with zigzag nano fabricated barriers for a DNA curtain experiment, connect the input and output tubes in the correct direction. Then wash the flow cell with 2.5 milliliters of lipid buffer using two three milliliter syringes, ensuring there is no bubble in the flow cell.

Remove the syringe from the output and inject one milliliter of the liposome solution three times with a five minute incubation between each injection. For healing, rewash the flow cell with 2.5 milliliters of lipid buffer slowly, and incubate at room temperature for 30 minutes. During the incubation, prepare the BSA buffer as mentioned in the text manuscript.

Then after 30 minutes of healing, wash the flow cell with 2.5 milliliters of BSA buffer from the output side. Next, inject 800 microliters of streptavidin buffer into the flow cell from the input side in two steps with a 10 minute incubation after each step. Then wash the flow cell with 2.5 milliliters of BSA buffer to remove all free streptavidin molecules.

Next, dilute the biotin Lambda DNA with cloned motifs using BSA buffer and inject it four times slowly at five minute intervals. During the injection, turn on the microscope in the scientific complimentary metal oxide semiconductor system. Then wash the tubing with 10 milliliters of double distilled water and rinse the prism and the tubing connector with water, 2%liquid cuvette cleaner, and 99%ethanol.

Next, draw up at least 20 milliliters of the imaging buffer into a 30 milliliter syringe. Set up the flow cell on the microscope stage and connect it to the microfluidic system. Using a flow rate of 0.03 milliliters per minute, flush the DNA molecules to the barrier for 10 minutes.

Then switch off the microfluidic system and incubate for 30 minutes. with the flow stopped, allowing the DNA to diffuse laterally. To image the EWS-FLI1 condensation formation on DNA curtains, open the imaging software and find and mark the positions of the nine zigzag 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 in 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. EWS-FLI1 molecules were visualized by detecting the mCherry labeled EWS-FLI1 signals obtained with a 561 nanometer laser.

The in vitro formation of EWS-FLI1 condensate at the site of the 25 GGAA repeats in the DNA substrate could be directly visualized. The specificity of the mCherry EWS-FLI1 used in DNA curtains was confirmed by an electrophoretic mobility shift assay using a DNA template with and without the 25 GGAA repeats separately. When the EWS-FLI1 concentration was titrated from 20 to 500 nanomoles the EWS-FLI1 intensity increased dramatically.

Whereas the change in the FLI one DBD intensity was negligible when the proteins were saturated to cover the GGAA repeats, suggesting that EWS-FLI1 formed condensates on DNA. The injection should be very slow and soft so that the liposome and DNA could distribute evenly on the flow cell. It is important to perform MSA to confirm that a purified transcription factor specifically bonds with target sequence in vitro.

This technique allows people to explore whether the condensate will facilitate the target search of transcription factors, and its effect on transcription.