Optical Tweezers Single Molecule Force-Extension

Contact: Jake Anna

Alumnus: Masha

In our lab, we exploit some distinct advantages of optical trapping to use it as a testbed for developing new single molecule spectroscopies:

  • The target molecule is localized between the two trapped beads to within a few nm, well below the diffraction limit
  • The target molecule sits in the focal point of a high NA microscope, which makes it straightforward to couple in probing light
  • The concentration of untrapped target molecules free in solution is nearly zero, due to the strong attachment to the beads, which leads to very low background levels even in the confocal geometry
  • The target molecule is far from any surfaces (e.g., glass slides or trapping beads), which further reduces optical background and undesired chemistry
  • The assay is non-perturbative, because it can be designed such that the trapping light is displaced from the position of the target molecule. Functional assays have shown that the attachment chemistry doesn’t disturb cofactor binding and hydrolysis, etc.
  • The target molecule’s orientation is restricted due to the topological restraints of the tethers although rotation about the extension axis is possible
  • Interesting chemical processes such as protein structural rearrangements and mechanochemistry can be triggered by force, and unsynchronizable, diffusion-limited processes can be observed in equilibrium.
  • Force-extension curves of PMMA tethers obtained using the optical trap. The data show in situ click chemistry linkages formed between (a) polymer-bead and (b) polymer-polymer. In each panel, five cycles are shown (extend, relax, extend...) that demonstrate force increase as the polymers are extended due to their entropic elasticity, and rupture in a single step (indicated by blue arrow). The dashed line indicates a worm-like chain fit. Solvents were THF and toluene, respectively. Traces are horizontally offset for clarity. Deviation from the fit at >35 pN arises from non-linearity of the trapping potential.
  • For single molecule spectroscopy in organic solvents, we have developed a click-chemistry analog to the antibody/antigen-linked dual bead construct.
  • The dual bead optical trap allows for a single molecule to be immobilized during force-extension experiments.
  • Masha, Anna, and Jake working at the optical trap