This protocol enables us to monitor the interaction dynamics and the resolution of the Holliday junction by flap endonuclease 1 at the single molecule level and other enzymatic systems. Well, some will averaging throughout the molecular population or secures the underlying individual mechanistic steps and there is. Single molecule methods provide these details by monitoring the real-time behavior of individual molecules.
Single molecule methods can efficiently detect biomolecular interactions in the pico and nanomolar range with high specificity. Which is handy for many practical solutions in diagnostics, genome sequencing, and sensing. Optical and forced measuring single-molecule methods are widely applied in Physics, Chemistry, and Biology and are proven to provide noble observations that are inaccessible by other methods.
My advice to first-time users is to closely follow the detailed steps laid out in this protocol. Visual illustrations of the hands-on procedure of this protocol will lead to successful implementation of the technique. To prepare the single channel flow cell, begin by drilling two 1.22 millimeter-diameter holes in the middle part of a 50 by 20 millimeter quartz slide with the centers 37 millimeters apart and 6.5 millimeters from the edge of the slide.
Use an electronic cutter to cut a 41 by 2.25 millimeter channel into a 50 by 20 millimeter piece of a double adhesive sheet and peel off the plastic side of the protective cover. Align the edges of the piece with the edges of the quartz slide and use a pair of polytetrafluoroethylene tweezers to gently remove any trapped air bubbles. Peel off the paper side of the adhesive piece and mount the piece onto the functionalized surface of a cover slip.
Next, cut one piece of polyethylene tubing with an internal diameter of 1.22 millimeters to an 11 centimeter length for the inlet and one piece of tubing to 25 centimeters for the outlet. Then insert the tubes into the previously drilled holes as the inlet and outlet tubes for the flow cell. And use five minute epoxy glue to seal the edges of the quartz cover slip interface in the inlet and outlet tubes.
When the cell has dried, use a 1 millimeter syringe to flush 0.2 micrometer filtered 0.03 milligram per milliliter concentration avidin in PBS into the cell using a second syringe filled with buffer alone to wash out any excess avidin at the end of the profusion. To perform a single-color FRET experiment, first apply one drop of immersion oil onto a 100 times total internal reflection fluorescence objective and set the on chip multiplication of photoelectrons to a suitable gain to optimize the signal to the background and to prevent saturation. Carefully place the flow cell onto the sample holder, and use course adjustment to gradually raise the objective until the oil touches the cover slip.
Turn on the 532 nanometer laser and switch to fine adjustment mode of the objective. Direct the emission to the camera port to observe the image on the monitor and adjust the height of the objective until the functionalized surface of the cover slip is brought into focus and can be observed on the monitor. Check that the background from the functionalized surface of the cover slips does not exceed few spots before flowing in the fluorescently labeled HJ.When the system is ready at 0.2 to 0.5 microliters of the diluted substrate of interest into 120 microliters of imaging buffer with oxygen scavenging system into a 0.5 milliliter tube and connect the outlet of the slow cell to a syringe pump.
Insert the inlet tube into the 1.5 milliliter tube and start the syringe pump at a 30 to 50 microliter per minute to withdraw the solution from the tube. Frequently image the surface briefly with the green laser to confirm a good coverage of the cell with 100 to 300 particles of homogeneously distributed, well-spaced substrate. If the flow cell surface is adequately covered, flush another 120 microliters of imaging buffer at 30 to 50 microliters per minute to wash away any unbound HJ and allow the flow cell to sit for 5 minutes to allow the oxygen scavaging system to deplete the dissolved oxygen.
Set the exposure time to approximately 60 milliseconds. The cycle time will be automatically set by the software based on the speed of data transfer. Specify the desired number of cycles or frames to approximately 400.
The emission from the donor and acceptor fluorophores are split into color channels by an image splitter device. Select a suitable area on the surface, adjust the height of the objective to focus the image and record and save the movie in 16 bit tiff format. Then move to a new area.
Flush the cell with 120 microliters of imaging buffer supplemented with the indicated concentrations of GEN1 at a 30 microliters per minute flow rate, one concentration at a time. In the resolution of the HJ cleavage experiment, adjust the flow rate to 110 microliters per minute and start the recording 5 to 10 seconds before the entrance of the enzyme into the flow cell. the imaging to start 5 to 10 seconds before the entrance of the enzyme into the flow cell will maximize the number of events.
When all the concentrations have been tested, use a fixed fluorescent bead slide to map the donor and acceptor particles to each other in the image-splitting device. After installing the appropriate software package to generate a transformation matrix, open the recorded bead slide movie and select the positions of the individual beads in the donor and acceptor channels. When the matrix has been generated from the fixed bead slide click Load TFORM and select the transformation matrix file.
In the channel filter drop down menu, click the D&A option to select for particles labeled with both the donor and acceptor. Then, enter plotTimetraceCW as instructed in the manual to generate the time traces for each molecule. To perform an ALEX FRET experiment, record a movie composed of consecutive frames of donor and acceptor emissions by direct excitations with the green and red lasers.
Open the acquired ALEX movies and set the suitable detection threshold at approximately 300 for each of the donor emission due to donor excitation, acceptor emission due to donor excitation, and acceptor emission due to the direct excitation channels. Apply the appropriate channel filters to select for the particles that have been both donor and acceptor and link 200 to 300 particles in each of the three channels. Use the plotHistALEX MATLAB code to generate ALEX histograms fitting different peaks in the histogram's functions and use a suitable graphing and analysis program to determine the percentage of the area under the curve for each population.
Then use the plotTimetraceALEX MATLAB code to generate a time trace for each molecule showing the donor emission by the direct excitation in the acceptor emissions due to the FRET and the direct excitation. This representative alternating excitation FRET histogram of adjacent label X-0 shows two peaks that correspond to interchanging of the more abundant and less abundant isomers. The isomerization rates obtained from the Dwell time histograms of the isomers is consistent with those reported previously.
Single molecule FRET histograms of the adjacent label X-0 junction at different GEN1 concentrations acquired by ALEX, reveal one peak corresponding to the free high FRET isomer and one peak corresponding to the bound HJ population after subtracting the contribution of the less abundant isomer from the low FRET peak. At a saturating GEN1 concentration, the FRET histogram of X-0 has only a single low FRET peak corresponding to GEN1 bound to either isomer of the HJ as predicted by the model. The binding of GEN1 monomer to the HJ followed by dimer formation is a unique feature of the eukaryotic HJ resolvase GEN1 compared to prokaryotic resolvases which exist in dimeric form in solution.
Further evidence that GEN1 monomer binds and distorts the HJ is the observation of a significant number of traces of uncleaved particles with a stable low FRET state in the presence of magnesium at low GEN1 concentrations. The simultaneous departure of the donor and acceptor after a stable low FRET state in traces of resolved X-0 that occurs without the emergence of an intermediate indicates that a complete resolution occurs within the lifetime of the GEN1 HJ complex. Note that the background from the functionalized glass surface should not exceed a few spots and that evenly distributed particles are essential for obtaining good results.
Always use personal protective equipment and a fume hood while preparing the serialization picture. Be sure to wear protective glasses specific to the wavelengths when working with lasers. Other methods such as cryo ap, nuclear magnetic resonance, can provide atomic level structural details.
This information are integral for the design and interpretation of the single molecule FRET experiments. Single molecule FRET open new views in the field of DNA replication resulting in deeper understanding of the mechanisms of actions helicases and nucleases.
