The dual DNA ruler assay can provide mechanistic insights for ribosome translocation and probe other nucleic acid displacements. Our dual DNA ruler assay is currently the only method that can objectively probe both the entrance and exit sides of mRNA in the ribosome complexes with single-nucleotide resolution. Similar methods are also applied to measure DNA binding strength and selectivity of chemotherapeutical drugs.
Visual demonstration of the method is a good chance for us to intuitively show the advantages of our technique and help other researchers to further develop it into more applications. To begin, make a plastic sample well with a styrene strip by drilling a cube in the center with dimensions of four millimeters by three millimeters by two millimeters. Then, using epoxy, glue a piece of biotin-coated glass, of approximately five millimeters long, to the bottom surface.
Add 20 microliters of 0.25 milligram per milliliter streptavidin aqueous solution into the sample well, and incubate at room temperature for 40 minutes. Then rinse the sample well twice with TAM10 buffer. To immobilize the ribosome complexes without antibiotics, first remove the buffer from the sample well, and add 20 microliters of 0.1 micromolar MF-Pre or MF-Post ribosome complex into the sample well.
Incubate at 37 degrees Celsius for one hour, and then rinse once with TAM10 buffer. The ribosome complex now binds with the streptavidin on the surface via the five-prime end biotin on the mRNA. For the experiment using antibiotics, incubate nine microliters of the MF-Pre complex with one microliter of four millimolar neomycin, 37 degrees Celsius for 10 minutes.
Then combine in a tube 0.6 microliters of 60 micromolar EFG, 0.8 microliters of 100 micromolar GTP, 0.8 microliters of 100 millimolar PEP, 0.3 microliters of 1.3 milligram per milliliter pyruvate kinase, 6.43 microliters TAM10 buffer, and one microliter of five millimolar fusidic acid. Incubate at 37 degrees Celsius for 20 minutes. Then combine the two mixtures, and incubate at 37 degrees Celsius for an additional five minutes.
Carry out the other antibiotics experiments similarly, with concentrations of 0.2 millimolar viomycin, 0.4 millimolar hygromycin B, and 0.25 millimolar fusidic acid. For frame shifting study, repeat the incubation for the MFNF-Pre complexes involving the slippery motif, U6A. Include antibiotics of fusidic acid plus neomycin.
Sufficient time is necessary to ensure the completion of the hybridization process. It is also advised to use the TAM10 buffer with actual one mole sodium chloride to maintain a homosteady system. After incubation, remove buffer from the sample well.
Add 20 microliters of one micromolar biotinylated probing DNA strand, and incubate at room temperature overnight. In the morning, rinse the formed DNA/mRNA duplex once with TAM10 buffer. Remove the buffer from the sample well.
Then add 20 microliters of 0.5 milligrams per milliliter streptavidin-coated magnetic beads into the sample well, and incubate at room temperature for two hours. After that, carefully insert the sample well into a holder, and place it in a centrifuge to remove the free magnetic particles from the surface at 84 times g for five minutes. Turn on the laser.
Then press the power button. Adjust the sensitivity to 500 millivolts, and wait for about two hours to warm up and stabilize the atomic magnetometer. To set up the atomic magnetometer, run the instrument control software, and set up the Motor moving mode to Noise and default position to zero.
Press Lock on the front panel. Adjust the sensitivity back to 200 millivolts. Adjust the current and voltage of the laser, and find the proper resonance peak and signal-to-noise level for the best signal resolution.
Press Sweep on the front panel. Make sure the amplitude-to-width ratio is above 0.5 and the phase value is smaller than five degrees with width less than 170 hertz. Plug in the output of another SR530 lock-in amplifier to the feedback of the laser to lock its frequency.
Plug in function generator ATF20B to input a square wave of 500 millivolts peak to peak, 100 millihertz as the reference signal for converting current to magnetic signal. Then unplug the function generator. Set up the Motor moving mode to Two-way and default position to 260 millimeters.
Check the status of the temperature controller, to ensure the temperature is at approximately 37 degrees Celsius, of the atomic sensor. Check the stability of the whole system twice by directly running the program without loading samples to measure the signals of the empty sample holder. To magnetize the samples, gently use tweezers to take the sample out of the holder, and place on the magnetization stage for two minutes.
Then put the sample back into the sample holder, and centrifuge at 335.4 times g for 20 minutes. Remove the nonspecifically-bound magnetic particles. Next load the sample onto the motor with tweezers.
Click Lock on the front panel to run the program. Meanwhile use tweezers to load another sample into the holder, and place it into the centrifuge. Make the coated glass side face the center of the centrifuge, and start the centrifugation at 335.4 times g for four minutes.
When the motor comes back to zero, after approximately five minutes, click Save on the front panel. Carefully use tweezers to transfer one sample from the motor to the centrifuge holder. Apply a stronger force by increasing the centrifugal speed by 100 rpm or a similar step size.
Exchange the sample for the centrifuge. Process alternately to gradually increase the force each time. A complete force spectrum is obtained after 10 to 12 data points.
When all planned experiments are finished, turn off the equipment, proceeding in the opposite order as it was turned on. Remove the samples from the holder, and immerse them in ethanol for cleaning and future use. Clean up the sample holder with acetone in case of magnetic bead contamination.
In this protocol, the ribosome complex was immobilized on the surface, via the five-prime end of the mRNA, and made both the five-prime and three-prime side easily accessible for the probing DNA rulers. When the linker for the five-prime side was longer than 50 thymine, a strong magnetic signal was detected, indicating successful formation of DNA/mRNA duplexes. By varying the number of nucleotides on the DNA, the correlation of dissociation force to duplex length was achieved for the three-prime and five-prime sides, respectively.
The results of normal translocation from MF-Pre to MF-Post, in the absence of antibiotics, show that the ribosome moved by three nucleotides toward the three-prime end on both sides. MF-Pre carried a vagrant fMet-tRNA and phenylalanine-tRNA at the P and A sites, respectively, while MF-Post carried fMet-tRNA and phenylalanine-tRNA at the E and P sites, respectively, with a vacant A site. However, when both fusidic acid and adiomycin antibiotics were present, the ribosome moved by only one nucleotide at the five-prime side but two nucleotides at the three-prime side.
After washing away the antibiotics, normal translocation occurred, as evidenced by three nucleotide movements from both the five-prime and three-prime sides. When magnetizing the sample, the coated surface should approach and leave the magnet vertically. And removing the nonspecifically-bound magnetic particles is very important for data accuracy and quality.
Our technique provides a high resolution and a generally applicable way to investigate molecular displacement of nucleic acid which is widely encountered in molecular biology.
