Koch Lab:Protocols/Optical Tweezers

Experiments

There are numerous experiments that could be performed using an Optical Tweezers. KochLab uses it for biological applications. And we separate the experiments into two regimes: DNA Applications and Kinesin Applications.

DNA Experiments

1. DNA Stretching - The essence of this technique is to stretch DNA. It is possible to label DNA so with biotin (for adhesion to a streptavidin coated microsphere) and digoxigenin (for adhesion to anti-digoxigenin). These labels allow for the creation of dsDNA tethers because of the nature of anti-dig bonding with glass. The microsphere allows our OT to probe the sample and stretch the DNA. From here there are many avenues to take. First you can reproduce worm-like chain analysis. You could also perform some protein-DNA analysis and observe the amount of force required to remove proteins bound to dsDNA. There are also experiments investigating the nature of DNA intercalaters enhanced by stretching.
2. DNA Unzipping - Steve created an unzipping construct allowing experimenters to unzip dsDNA. This works in large part because of how DNA basepairing works, how most restriction endonucleases cleave DNA, and thanks to ligation (attaching 2 dsDNA sequences together). Embedded in the unzipping construct is a nick along the sugar backbone which allows dsDNA to be pulled apart at the site of the nick. Just slightly downstream of the nick is a biotinylated nucleotide (again to be bound to a streptavidin coated microsphere) where forces from the tweezers will be applied. This construct is extremely versatile because it can be ligated to anything with a specific overhang. The sequence for this overhang can be found in a lot of popular plasmids. Currently KochLab is using this setup for genetic mapping which we call Shotgun DNA/Chromatin Mapping (SDM/SCM). Anthony also believes that this setup can be used for Telomere mapping.
3. Transcription - With careful consideration, RNA Polymerases can be affixed to a surface. If said Polymerase is in the midst of transcription, one could gain insight into the mechanism of this process. By using some form of one of the above techniques OT can be used to gain the insight. With a biotinylated DNA molecule being transcribed, OT can probe forces involved in elongation. If the setup is more like unzipping, studies can be done to understand binding of polymerases to DNA during different junctures of transcription.

Kinesin Experiments

1. Bead Motility Assay - Kinesin stalk/tail can be labeled with biotin so that a streptavidin coated microsphere can be attached to the molecular motor. In this experiment microtubules get affixed to a surface. Kinesin has been shown to walk along these MTs via ATP hydrolysis. With OT you can directly observe the motion of one kinesin walking along a MT via the microsphere (which is bound to a kinesin stalk). More advanced experiments have shown direct observation of each kinesin motor domain (the head groups) by genetically engineering the heads. The heads were engineered with short DNA oligos attached and biotinylated ends for microsphere adhesion. Other experiments lie on the horizon.