This protocol provides a high throughput technique to study the intermolecular binding between DNA and ligands. This technique has demonstrated high throughput in the investigation of massive set of biomolecular structures. It does so by converting the mechanical chemical properties of individual molecules into those of molecular ensembles.
I think it is a very step forward experiment but there are few critically steps. For example, the alignment of microscope, homogenizer, and the reaction tube. These three things should be perfectly aligned in order to accurately track the change in fluorescence.
Begin by assembling the homogenizer and the microscope on a mounting table. After wearing goggles, turn on the fluorescence microscope and then adjust the homogenizer to make sure that the excitation light beam passes through the center of the dispersing tip of the homogenizer. Then prepare a flat bottomed reaction chamber that is five centimeters in height and 1.5 by 1.5 square centimeters in cross section, ensuring that the selected chamber material does not fluoresce to reduce the background.
Afterward, clamp the reaction chamber on the sample stage of the fluorescence microscope and then adjust the chamber to hold the dispersing tip of the homogenizer, ensuring that the tip is slightly above the bottom surface of the reaction chamber. Next, adjust the vertical position of the homogenizer and the chamber together to ensure the focus of the microscope is on the surface of the dispersing tip. Then adjust the horizontal position of the dispersing tip to ensure that the detection area is set between the rotor and stater.
Switch on the fluorescent channels, according to the fluorescent dye used in the experiment. Before the high speed shearing experiment, used deionized water to test the shearing with a low shearing speed. First, prepare a human telomeric i-motif DNA labeled with a dye and a quencher at its two ends respectively in deionized water as described in the manuscript.
Next, dilute the DNA to five micromolar in 30 millimolar MES buffer at pH 5.5 or pH 7.4. To the DNA solution, add ligand L2H24OTD"in a concentration range of zero to 60 micromolar and mix the solution gently for 10 minutes to fold the i-motif structures without light. Check and minimize the background fluorescence intensity of the reaction chamber that is filled with deionized water using the fluorescent microscope without shearing.
Then set the parameters of the CCD camera using the software with exposure time as 0.5 seconds, CCD sensitivity of 1, 600, and recording time equal to 20 minutes. Using a long pipette, add the DNA solution into the empty and clean reaction chamber and cover the reaction chamber with a black box. Then start the homogenizer to perform shearing at a selected shear rate ranging from 9, 724 per second to 97, 245 per second for 20 minutes, with the CCD camera turned on to record the data.
After the experiment, remove the chamber and wash it with deionized water. Prepare i-motif DNA and deionized water. Then, incubate 10 micromolar i-motif DNA and 300 microliters of 30 millimolar tris buffer supplemented with 150 micromolar cupric chloride and 300 micromolar ascorbic acid for 10 minutes to fold the i-motif structures.
Next ultra filtrate the solution with an ultra filtration device at a centrifugal force of 14, 300 times G.Replenish the solution to approximately 500 microliters with 30 millimolar tris buffers supplemented with 300 micromolar ascorbic acid after each filtration. After collecting the residual solution, make a final volume of 300 microliters by adding 30 millimolar tris supplemented with 300 micromolar ascorbic acid along with 20 micromolar CalFluor 488 Azide, 20 micromolar HPG, and 10 micromolar TBTA. Once the reagents are added, move the solution to the dark room.
Then check and minimize the background fluorescence intensity of the reaction chamber filled with deionized water using the microscope before the shearing experiments. Next, add the DNA solution into the empty reaction chamber with a long pipette, and then start the homogenizer shearing at a shear rate of 63, 209 per second for 20 minutes with the CCD camera turned on. After the experiment, remove the chamber and wash it with deionized water.
The fluorescence intensity of i-motif DNA was observed to increase with the sheer rate ranging from 9, 724 per second to 97, 245 per second in a pH 5.5 MES buffer. However, fluorescence intensity was not increased when the same i-motif DNA was sheared at a rate of 63, 209 per second, as it does not fold at pH 7.4 in MES buffer. The ligand binding to the DNA i-motif molecules subjected to the sheer flow was evaluated which showed that the increment of the fluorescence intensity became less obvious with increasing ligand concentration.
The dissociation constant was determined for the interaction between the ligand and the i-motif and was found to be 34 micromoles. The copper one chelated i-motif was unfolded at the 63, 209 per second shear rate, and the released copper one triggered the fluorgenic click reaction, resulting in increased fluorescence intensity over time. Firstly, make sure that the detection area is set between rotor and the stater.
Second, check and minimize the fluorescence background. And lastly, make sure you filter the reaction before you perform click reaction. This method opens a gateway to explore mechanical strength, folding and unfolding off all kinds of polymers.
It could be other DNA structures, proteins, or other polymers, and their interaction with other kinds of molecules. As said, several other macromolecules could be studied using this approach.
