Single-Molecule Imaging of Fully Reconstituted CMG with Correlative Dual-Beam Optical Tweezers and Confocal Microscopy

Perform the single molecule imaging in a commercial setup, combining dual beam optical tweezers with confocal microscopy. Use inject channel one for bead trapping, channel two for DNA protein complex binding, channel three for checking the presence of CMG and channels four and five as protein reservoirs and buffer exchange locations. Flow all solutions into the flow cell at a constant pressure of 0.5 bar in the injection port.

Then turn off the flow in channels four and five. After initially flowing all solutions, reduce the pressure to 0.2 bar. Move trapping lasers to channel one until one bead is caught in each optical trap.

Move trapped beads to channel two by moving the joystick while pressing the trigger. Using the joystick without pressing the trigger fish a DNA CMG complex by moving the right bead towards and away from the left bead until a DNA tether is trapped. Move the beads to channel three and immediately stop the flow in all channels.

Adjust the 561 nanometer laser power to four microwatts in the excitation laser panel and then take a one frame test scan of the DNA in channel three. If present, CMG will appear as two dimensional diffraction limited spots. In this case, move the DNA tether to either channel four or five for imaging.

Otherwise, turn the flow back on, discard the beads and repeat the steps. In channels four or five input two piconewtons in the force spectroscopy panel, and click on the enable button to start a force clamp. Once the initial tension reaches two piconewtons, disable the force clamp before imaging by clicking again on the enable button in the force spectroscopy panel.

Click the start scan button in the image scan panel. Image fluorescent CMG every five seconds with a 561 nanometer laser at a power of four microwatts as measured at the objective. In an aggregation free reaction, CMG appeared as discrete, symmetrical diffraction limited spots sparsely crowding the DNA.

On the contrary, aggregates are less discrete, sometimes asymmetrical blobs crowding a larger length of the DNA. If the assay was successfully executed and high purity of the purified proteins was achieved, long range motion of CMG in the presence of ATP could be observed.