Quantifying Molecular Forces with Serially Connected Force Sensors

To understand the mechanical forces involved in cell adhesion, molecular force sensors have been developed to study tension through adhesion proteins. Recently, a class of molecular force sensors called tension gauge tethers (TGTs) have been developed that rely on irreversible force-dependent dissociation of a DNA duplex to study cell adhesion forces. Although the TGT offers a high signal-to-noise ratio and is ideal for studying fast/single-molecular adhesion processes, quantitative interpretation of experimental results has been challenging. Here, we use a computational approach to investigate how TGT fluorescence readout can be quantitatively interpreted. In particular, we studied force sensors made of a single TGT, multiplexed single TGTs, and two TGTs connected in series. Our results showed that fluorescence readout using a single TGT can result from drastically different combinations of force history and adhesion event density that span orders of magnitude. In addition, the apparent behavior of the TGT is influenced by the tethered receptor-ligand, making it necessary to calibrate the TGT with every new receptor-ligand. To solve this problem, we proposed a system of two serially connected TGTs. Our result shows that not only is the ratiometric readout of serial TGT independent of the choice of receptor-ligand, it is able to reconstruct force history with sub-pN force resolution. This is also not possible by simply multiplexing different types of TGTs together. Last, we systematically investigated how the sequence composition of the two serially connected TGTs can be tuned to achieve different dynamic range. This computational study demonstrated how serially connected irreversible molecular dissociation processes can accurately quantify molecular force and laid the foundation for subsequent experimental studies.