Biomolecules undergo small and rapid structural changes often in response to force. High resolution magnetic tweezers can explore these dynamics under physiologically relevant forces. As the method makes nanoscale measurements with millisecond precision, it is possible to monitor subtle changes in nucleic acids and proteins in real time.
Malfunctions in first bearing and mechanosensitive proteins can potentially lead to cardiovascular and musculoskeletal disorders. Magnetic tweezers can provide insight on the working mechanisms of these proteins. The set must be properly aligned and calibrated to obtain good resolutions.
Also, care must be taken during the preparation and identification of molecular constructs for measurements. Begin by setting up an inverted microscope on an anti-vibration optical table. Next, install a high speed CMOS camera and a frame grabber.
Vertically mount a motorized linear stage with more than 20 millimeter travel length on a manual XY stage to build a translation stage for magnet manipulation in 3D. Next, install a rotary stepper motor and a belt and pulley system for magnet rotation. Mount the magnets on an acrylic holder in which the identical parallel magnets are spaced one millimeter apart.
Adjust the vertical position of the translation stage so that the magnet's bottom surface aligns with the sample plane at the lowest stage position. Using a low magnification objective lens, align the magnets to the center of the field of view. Check the magnet rotation to ensure that the displacement of the center of the magnet pair is limited.
Install a super luminescent diode for bead illumination. Pass the beam through the magnet gap ensuring the beam is properly collimated to fit in the gap and the illumination is not shadowed by the magnets. Now install a Piezo lens scanner on the nose piece and mount 100X oil immersion objective lens with a 1.5 numerical aperture for bead tracking.
Ensure that illumination is uniform with magnet movement to avoid potential artifacts in bead tracking. Finally, adjust the light to maximum brightness without pixel saturation. Begin by taking two glass coverslips for the top and bottom of the cell.
Clean the coverslips by sonicating them in one molar potassium hydroxide for 30 minutes. Next, rinse the coverslips in distilled water and keep them immersed in water. Pegylate the bottom coverslip and store it at minus 20 degrees Celsius until further use.
On the day of the experiment, blow dry the pegylated coverslips with a nitrogen gun. To make the simple channels, prepare approximately two millimeter wide strips of double-sided tape and lay down four strips parallel to each other five millimeters apart on the bottom coverslip. Now place a top coverslip in the center of the bottom coverslip leaving about five millimeter space on the short edges for channel inlets and outlets.
Using tweezers, gently press the back of the top coverslip to firmly seal the channels. Trim the edge of a 200 microliter pipette tip and cut out about 10 millimeters from the wider opening to allow it to hold more than 200 microliters of solution. Prepare three syringe needles that fit the tubing for the syringe pump.
Connect the needle on the flow cell channel exit to the syringe pump with polyethylene tubing. Equilibrate the channels with PBS. Resuspend bead aggregates by vortexing the bead solutions.
Then sequentially introduce the required solutions into the channel by suctioning with the pump. Wash away any unbound beads while applying 0.1 piconewtons of force. On the flow cell channel surface, identify the magnetic beads tethered to single molecules of the DNA construct.
Locate a reference bead nearby. Rotate the candidate bead to check if it swivels freely. Rotate the beads for a few more turns and estimate the radius of rotation, choosing a bead with a smaller rotational radius.
Increase the force from zero to five piconewtons to identify good single tethered beads by looking for a large change in the bead diffraction pattern resulting from a five kilobase pair tether stretch. Using the PCR, prepare five kilobase pair of double-stranded DNA fragments labeled with biotin on one end and azide on the other end. After preparing a flow cell with the PCR product, record the X and Y coordinates of the tethered bead at 1.2 kilohertz and the magnets in the resting position.
Move the magnets closer to the flow cell and repeat the bead position measurements until the magnets barely touch the top of the flow cell. Calculate the force at each magnet position D using either of the alternative methods. Repeat the force measurements for a few more DNA constructs probing three to five beads to average out the force variability between the magnetic beads.
Once a proper magnetic bead is located together with a reference bead, click on the calibrate button to start preparing for bead tracking. Click on the beads in the image to define the locations of the beads. For tracking in the Z direction, the software will step the objective lens with a Piezo scanner in equidistant steps and record fluctuation averaged bead images at each position to generate a lookup table.
Enable tracking and autofocusing and click on the acquire button to record the bead positions. Force application to a DNA hairpin construct resulted in the worm-like chain model of force-induced extension. At six piconewtons, the construct displayed fluctuations in extension associated with reversible unzipping, which disappeared at eight piconewtons and snapped onto a new model curve.
Hairpin tracking experiments at 100 hertz showed an increase in extension, but the histograms did not resolve distinct populations. At 1.2 kilohertz, the median filtered trajectories revealed two distinct populations. The separation between the two populations remained the same in the unzipping force regime.
The upper open state became gradually dominant with increased force. Transition rates varied exponentially with applied force favoring unzipping and inhibiting rezipping. In the intermediate force regime, an Allan deviation of two to three nanometers was obtained at the maximum speed.
The DNA handles of a SNARE complex construct behaved as worm-like chain polymers. When the force was relaxed from a higher regime, the extension returned to the original curve or followed a new model. At 14 piconewtons, the SNARE complex failed to transition to the unzipped state, whereas an increased force allowed the transition.
The complete unfolding of the complex at higher forces was confirmed by refolding observed at two piconewtons. Identification of properly formed sample bead tethers is the most important thing to remember when attempting our procedure. This step enables accurate selection and thus analysis of the target structure.
Magnetic tweezers can further be used to study DNA super coiling by applying torque. Also, it could be coupled with fluorescence imaging to observe highly complex protein interactions or unfolding.
