This protocol shows how DNA curtain technique is utilized for studying chromatin dynamics, in particular investigating the molecular function of histone chaperones. High-throughput single-molecule imaging technique DNA curtain overcomes limitations of bulk experiments and facilitates Real-Time imaging, enabling the detailed characterization of Biomolecule interactions. Put a clean paper of dimension 5 by 35 millimeters on the center of the double-sided tape.
Attach the tape over the slide to cover the two holes and nano patterns with the paper. Then excise the paper using a clean blade. Put a glass cover slip on top of the double-sided tape, and rub the cover slip using a pipet tip to form a microfluidic chamber.
Place the assembled flow cell between two microscope slides, and clip them. Bake the flow cell in a 120 degrees Celsius vacuum oven for 45 minutes. Attach the fluid connector for the chip-based analysis to each open hole using a hot glue gun.
And connect the two lines of lure lock tubing. Connect a lure lock syringe containing three milliliters of deionized water, and wash the chamber. Wash the chamber again with three milliliters of lipid buffer via drop-by-drop connection.
Inject one milliliter of 0.04x biotinylated lipid in lipid buffer, into the chamber, in two shots, with a five minute incubation per shot. Inject 0.025 milligrams per milliliter of streptavidin in one milliliter of BSA, with two shots, and a 10-minute incubation per shot. Inject approximately 30 picomolar of biotinylated lambda phage DNA in one milliliter of BSA buffer, into the chamber with two shots, and 10-minute incubations per shot.
Connect a syringe containing 10 milliliters of imaging buffer to a syringe pump, and remove bubbles in all tubing lines in the microfluidic system. Couple the prepared flow cell with the microfluidic system via drop-to-drop connection to avoid bubble injection into the flow cell. Assemble the flow cell and flow cell holders, and mount the assembly on a custom built, total internal reflection fluorescence microscope.
Inject 200 microliters of YOYO-1 dye in imaging buffer through 100 microliters of sample loop to stain Lambda DNA molecules. Run imaging software, and turn on the 488-nanometer laser to check whether DNA curtains are well-formed. After YOYO-1 dye has been removed, inject the pre-incubated protein sample.
When the proteins reach the DNA curtain, turn off the syringe pump, switch off the shutoff valve, and incubate for 15 minutes for histone loading by Abo1. Wash out the unbound Abo1 and histone proteins for five minutes. Transiently turn off the buffer flow to check if histone proteins are loaded onto DNA.
DNA curtain formation was checked by staining DNA molecules with an interpolating dye. Green lines shown in parallel arrays indicated that YOYO1 intercalated into DNA molecules were well aligned and stretched at a diffusion barrier under hydrodynamic flow. When Cy5-H3-H4 dimers were injected into the DNA curtain in the absence of Abo1, Cy5-H3-H4 did not bind to DNA, indicating a lack of spontaneous binding of H3-H4 dimers to DNA.
When Cy5-H3-H4 was injected with Abo1, red fluorescent puncta were seen on DNA molecules, suggesting that Abo1 loads H3-H4 dimers onto DNA. The fluorescent signals disappeared in the absence of buffer flow, and reappeared when the flow was resumed, suggesting that H3-H4 dimers bind to DNA. The binding of H3-H4 dimers to DNA was also confirmed by the kymograph, in which the fluorescence signals disappeared whenever the flow was turned off.
Few fluorescent puncta appeared in the DNA curtain, either in the absence of nucleotide or in the presence of ADP, indicating that H3-H4 dimers rarely bind to DNA, either in the Apo state or in the presence of ADP. The quantitative analysis shows that the number of H3-H4 dimers bound to DNA increases in the presence of ATP. The results suggest that ATP hydrolysis is essential for H3-H4 loading onto DNA by Abo1.
Avoiding bubble injection into flow cell is the most critical for all steps. So in this protocol, we emphasize drop-by-drop connections. DNA curtain assay can be extended to double-tether DNA curtains with dual nano-patterns.
In addition, we can make another type of DNA curtain with single stranded DNA, RNA, or actin filaments. DNA curtain enables visualization of protein motion on DNA and facilitates studying on target search mechanism. DNA curtain can be extended for studying DNA metabolism including replication and transcription.
