In silico analyses of molecular force sensors for mechanical characterization of biological systems.

Mechanical forces play key roles in biological processes such as cell migration and sensory perception. In recent years, molecular force sensors have been developed as tools for in situ force measurements. Here, we use all-atom steered molecular dynamics simulations to predict and study the relationship between design parameters and mechanical properties for three types of molecular force sensors commonly used in cellular biological research: two peptide and one DNA based. The peptide-based sensors consist of a pair of fluorescent proteins that can undergo Förster resonance energy transfer, linked by spider silk (GPGGA) or synthetic (GGSGGS) disordered regions. The DNA-based sensor consists of two fluorophore-labeled strands of DNA that can be unzipped or sheared upon force application with a Förster resonance energy transfer signal as readout of dissociation. We simulated nine sensors, three of each kind. After equilibration, flexible peptide linkers of three different lengths were stretched by applying forces to their N- and C-terminal Cα atoms in opposite directions. Similarly, we equilibrated a DNA-based sensor and pulled on the phosphate atom of the terminal guanine of one strand and a selected phosphate atom on the other strand for pulling in the opposite direction. These simulations were performed at constant velocity (0.01-10 nm/ns) and constant force (10-500 pN) for all versions of the sensors. Our results show how the force response of these sensors depends on their length, sequence, configuration, and loading rate. Mechanistic insights gained from simulations analyses indicate that interpretation of experimental results should consider the influence of transient formation of secondary structure in peptide-based sensors and of overstretching in DNA-based sensors. These predictions can guide optimal fluorophore choice and facilitate the rational design of new sensors for use in protein, DNA, hybrid systems, and molecular devices.