High throughput mechanotyping via flow cytometry and DNA sequencing

Cells exert force on their surrounding environment, these forces contribute to proper cell development and health. Recent work has demonstrated that dysregulation of these forces plays a role in many disease states, including cancer growth and metastasis. Unfortunately, measuring the forces cells exert traditionally requires high-resolution but low throughput imaging of surfaces and/or arduous experimental preparation of materials and surfaces. To address these challenges, we have developed Rupture and Deliver Tension Gauge Tethers (RAD-TGTs), a high-throughput, a facile method to relatively measuring the force a cell exerts, allowing for the determination of a cells mechanical phenotype (mechanotype). RAD-TGTs consist of DNA duplexes conjugated to a ligand and indicator (fluorophore, barcode, etc.) which rupture in a force-dependent manner when cells are bound and exert a critical force. Following rupture, the bound oligo is delivered to the cell and the indicator allows for quantification of rupture events through flow cytometry or DNA sequencing. Additionally, we leveraged nucleases that form DNA-Protein covalent adducts to conjugate the ligand of interest to the unmodified duplex in a single-step process, which greatly simplifies the preparation of tension sensors. We show here that RAD-TGTs can rapidly profile a cell's mechanotype as demonstrated by a decrease of fluorescent signal following force-reducing drug treatment and knockout of proteins that contribute to mechanotransduction, highlighting the function of RAD-TGTs to be integrated into CRISPR screens. Furthermore, we demonstrate the ability to monitor progression Intriguingly, we demonstrated that RAD-TGT rupture can be quantified via DNA sequencing bringing cellular force measurements into the -omics era.