Forces play a fundamental role in a wide array of biological processes, regulating enzymatic activity, kinetics of
molecular bonds, and molecular motors mechanics. Single molecule force spectroscopy techniques have enabled the
investigation of such processes, but they are inadequate to probe short-lived (millisecond and sub-millisecond) molecular
complexes. We developed an ultrafast force-clamp spectroscopy technique that uses a dual trap configuration to apply
constant loads to a single intermittently interacting biological polymer and a binding protein. Our system displays a delay
of only ∼10 μs between formation of the molecular bond and application of the force and is capable of detecting
interactions as short as 100 μs. The force-clamp configuration in which our assay operates allows direct measurements of
load-dependence of lifetimes of single molecular bonds. Moreover, conformational changes of single proteins and
molecular motors can be recorded with sub-nanometer accuracy and few tens of microseconds of temporal resolution.
We demonstrate our technique on molecular motors, using myosin II from fast skeletal muscle and on protein-DNA
interaction, specifically on Lactose repressor (LacI). The apparatus is stabilized to less than 1 nm with both passive and
active stabilization, allowing resolving specific binding regions along the actin filament and DNA molecule. Our
technique extends single-molecule force-clamp spectroscopy to molecular complexes that have been inaccessible up to
now, opening new perspectives for the investigation of the effects of forces on biological processes.