Feedback Controlled Optical Tweezers for Measuring Lambda Exonuclease Activity

A single molecule is enough to initiate a biological function. Therefore methods to study single biomolecules are important. By measuring one biomolecule at a time it is possible to reveal the differences from molecule to molecule. Moreover, with these methods one can measure short-lived non-equilibrium states of the molecules. An optical tweezers instrument for single molecule experiments was modified to permit constant force experiments. The resulting force-clamp control was then used to measure enzymatic activity of the molecular motor lambda exonuclease at different temperatures.

The force-clamp experiments were performed in a dumbbell geometry where two polystyrene spheres are connected by a DNA molecule. A single continuous wave laser source created the dual trap optical tweezers with one stationary and one steerable trap. Acousto-optic deflectors steered the trap according to a digital feedback control from a field programmable gate array. Using the feedback control, at 1.3±0.3 pN force, the lambda exonuclease reaction rate was measured to be 23±16 nt/s at 25±1°C and 54±15 nt/s at 37±1°C.

The most important achievement in this thesis was the detected increase in the reaction rate of lambda exonuclease due to the increased temperature. Thus, the real-time force- clamp control opens up new possibilities to study the molecular machinery that generates forces and movements in living organisms.