Abstract Mechanical forces play key roles throughout biology, from governing the adhesion of leukocytes in the immune response, to determining cell fate and directing tissue formation. This field of mechanobiology is providing vital insights into conditions such as bleeding disorders, cancer, and infectious diseases, where it is becoming clear that conventional biochemical and genomic characterizations are not sufficient to understand the rich behavior of living systems or how they fail. Rather, we must uncover how force changes the structure and function of molecules, triggering mechanotransduction pathways to modify cell responses. Technological developments that enable precise manipulation of single molecules and cells have been a driving force in the development of the field, but growth has been impeded by both limited access to such technologies and by constraints in their capabilities, which has restricted the types of scientific questions that can be addressed. To overcome these challenges, we will develop approaches in mechanobiology that will (i) open up new areas of study through the introduction of new capabilities, and (ii) democratize single-molecule and nanoscale methods so that all biomedical researchers can make discoveries using these powerful tools. We will continue to develop instruments such as the Centrifuge Force Microscope, a miniature microscope that fits into a benchtop centrifuge to enable even non-specialists to perform high-throughput single-molecule force measurements, and nanoscale devices such as programmable DNA nanoswitches. We will develop DNA nanoswitch calipers, a tool capable of measuring distances on single-molecules with angstrom-level precision to enable single-molecule protein identification and shape determination. We will also develop Functional Interaction-based Nanoswitch Discovery (FIND), a high-throughput screening assay based not on traditional robotics, but on molecular devices that bring together molecular components to analyze and screen for interactions of interest. FIND will enable screening of complex modes of action to find compounds that activate a specific downstream pathway or allosterically stabilize a particular protein conformation. We will apply our nanoscale approaches to answer key open questions in mechanobiology. For example, to uncover the mechanical regulation of hemostasis we will use single-molecule methods to study the force- regulated enzymatic cleavage of von Willebrand factor, and the flow-induced elongation and activation of its adhesive function. We will also investigate the molecular basis of hearing and deafness by using single- molecule force spectroscopy to probe the properties of the hair cell tip link, and combine this approach with single-channel conductance measurements to simultaneously measure the force required to open mechanotransduction channels and the molecular movements that underlie channel gating. Overall, these efforts should firmly establish force as a...