This protocol is significant because it enables the utilization of existing cell-free translation systems for the single molecule analysis of active eukaryotic translation. The main advantages of this technique are that it preserves bulk translation properties, provide single molecule resolution, and is relatively simple to adapt from other bulk translation assays. This method can be applied to any cell-free translation system that has been verified to enable efficient cap-dependent translation under bulk conditions.
Demonstrating the procedure will be Hongyun Wang, a research associate, and Anthony Gaba, a senior research scientist from Xiaohui Qu laboratory. At least three hours before performing a single molecule experiment, turn on the microscope incubator and reduce the airflow to a low rate to avoid excessive acoustic disturbance of the experiment. Then set the incubator temperature to the optimal temperature specific for the cell-free translation system.
At least one hour before the experiment, press the laser power button behind the laser box, turn the laser safety key, and press the start button to turn the laser on. Turn on the microscope and select the imaging mode, objective, and filter set. Turn on the EMCCD camera, the stage controller, the syringe pump and the computer and open the camera, laser, motorized stage, and syringe pump software.
In the laser control software, click connect to set the laser intensity, uncheck the shutter box and click control to drag the intensity slide bar in the laser control pop-up window. Enter the penetration depth to set the laser to the desired angle and confirm that the shutter box is closed to maintain the laser in the off mode when not imaging. When the camera has cooled and stabilized to its designated working temperature, in the camera software, select kinetic for the acquisition mode and enter the exposure time, kinetic series length, and electron multiplying gain value parameters.
Select the directory and assign filenames for saving the data image file. Set the syringe pump to a modest to fast flow rate and add 500 microliters of 70%ethanol to a microfuge tube. Insert the syringe pump tubing end into the ethanol solution and use the syringe pump software to toggle the syringe pump between the withdrawal and dispense modes to wash the tubing three times with ethanol and three times with RNase-free water.
After the final water wash, dab the tubing end on a clean tissue wipe to remove any residual water from the inside of the tubing and place the end of the tubing into a clean microfuge tube. For three prime end biotinylated mRNA immobilization, place the flow chamber into the microscope sample holder with the coverslip side facing down and secure the chamber with tape. Use the tape to cover the flow channel inlets and outlets that are not in use and add 1.5 times the volume of a 200 microgram per milliliter streptavidin solution to the flow channel.
After 10 minutes at room temperature, wash and wipe the channel three times with three times the volume of T50 wash buffer per wash and the folded laboratory tissue. After the last wash, place a drop of immersion oil onto the microscope objective and place the holder and chamber onto the microscope stage. Then bring the objective close to the coverslip until the immersion oil on the objective contacts the coverslip and move the sample stage so that the position of the flow channel is directly above the objective lens.
On the microscope control touchscreen, select the focus tab and click focus search and start. A beep will be heard when the focus plane is successfully achieved. In the camera software, select the live mode.
In the laser control software, open the laser shutter and adjust the laser intensity level. Observe the real-time fluorescence image in live mode and in the microscope control touchscreen using the fine step control to adjust the objective position. In the camera software, move the stage to evaluate the level of fluorescence background at various areas of the flow channel detection surface.
If the fluorescence is low, stop the live mode and close the laser shutter in the laser control software and replace the wash buffer from the flow channel with two times the volume of the biotinylated mRNA solution. After 15 minutes, wash the channel three times with three times the volume of translation compatible buffer per wash. For single molecule detection, assemble three times the volume of freshly thawed translation mix without mRNA and supplemented with an optimized concentration of fluorescently labeled antibodies in a microcentrifuge tube on ice and briefly spin the tube to collect the mix at the bottom of the tube.
Transfer the translation mix to the inside of a 700 microliter microfuge tube cap and set the syringe pump to the withdraw mode. Insert the pump tubing end into the translation mix and start the syringe withdrawal. When the translation mix has completely entered the pump tubing, stop the withdrawal, reverse the syringe pump settings to the dispensing mode, and set the flow to an optimal rate for translation mix delivery.
Start and stop the syringe to allow the translation mixture to reach end of tubing as necessary and insert the pump tubing end into the inlet of the flow channel, taking care to avoid bubbles. Screw the custom-made metal bracket onto the microscope sample holder plate to stabilize the connection and assess the microscope focus and imaging area fluorescence. Confirm that the movie acquisition parameters are correct and that the appropriate filename and directory have been entered for saving the files.
Move the stage to allow imaging of an area in the center of the flow channel detection surface. And under the continuous AF tab, click the start buttons. A beep will be heard when a focus plane has been successfully achieved.
After reducing the ambient light, open the laser shutter and click the take signal button to start recording. After five seconds, enter the run command to deliver the translation mix into the flow channel. Then record the data for up to two hours at a 0.5 to two seconds per frame time resolution.
To analyze Cy3 ANTI-FLAG binding events, the fluorescence intensities of selected Cy3 alpha flag spots were calculated frame by frame for the entire movie and connected to form an intensity trajectory. Raw trajectories can appear noisy and may require refinement with a non-linear forward/backward filter to reduce background noise. Further refinement can be achieved using step detection algorithms to digitize trajectories.
Antibody binding to a nascent polypeptide causes an instantaneous increase in fluorescence, whereas antibody polypeptide dissociation causes an instantaneous fluorescence decrease. The dwell time histogram for luciferase flag mRNA fits to a log normal distribution and yields a peptide synthesis rate of 2.5 plus or minus 0.1 amino acids per second. The first arrival time histogram fits to a shifted log normal function, illustrating the high degree of heterogeneity in single mRNA translation activity.
As the histogram indicates, the first peptide synthesis event on single luciferase flag mRNA molecules can begin as early as two minutes and as late as 20 minutes after translation mix delivery. Compared to translation with luciferase flag mRNA, the first arrival time histogram with hairpin luciferase flag mRNA translation showed slower antibody binding. Using bulk luminescence measurements from cell-free translation reactions with these same mRNAs, however, does not resolve differences in translation kinetics.
The single molecule resolution of this assay provides more kinetic insights of cap-dependent translation that are not easily attainable with other approaches. Eukaryotic cell-free translation is sensitive to small variations and experimental conditions. Therefore, attention to detail and consistency are critical when using this assay to accurately detect small differences in translation.
