We have built an axial optical tweezers to stretch sub-micron DNA constructs and study protein-mediated DNA loop formation and breakdown rates in an in-vitro model based on the lac repressor of E. coli. A section of DNA carrying two LacI-binding operators with an inter-operator spacing of a few hundred basepairs is attached by one end to a microscope cover glass while, at the other end, a microsphere serves as a handle for the optical tweezers.
The microsphere is trapped in the approximately linear region of the optical potential in the axial direction, where the optical force is almost independent of the axial position of the microsphere. This way we are able to directly stretch the molecule in the axial direction by increasing the laser intensity. Loop formation and breakdown is observed as an apparent change in the length of the stretched DNA. The tension in the DNA is carefully calibrated, accounting for small yet significant volume exclusion forces, then quantitatively determined from the extension of the DNA molecule using the wormlike chain model.
We observe that when the tension increased by about 100 fN, the loop formation rate increases by an order-of-magnitude, whereas the loop breakdown rate remains virtually unchanged. This result can be discussed in the context of a theoretical model for protein-mediated DNA looping under tension that allows us to infer loop topology from the force sensitivity data. Our experimental results demonstrate that forces as low as hundred femtonewtons can indeed have a dramatic impact on the formation of regulatory protein-DNA complexes, suggesting possible mechanical pathways to gene regulation.
Looping lifetime as a function of applied tension. By applying only a few hundred femtonewtons of force, we can greatly increase the time it takes to form a loop, which essentially means that tension could act as a genetic switch
