Tension tolerance of TGT

Tension gauge tethers (TGTs) with 18 bp DNA duplex region were used¹⁹. The sequence of duplex region is GGC CCG CAG CGA CCA CCC (ligand conjugated strand). The nominal values of TGT tension tolerance were used as estimated by previous reports^11,19. In brief, DNA rupture force was estimated based on the model formulated by de Gennes: Ttol=2fc[κ−1tanh(κl/2)] where f_c is breaking force per base pair (3.9 pN), l is the number of base pairs, and κ⁻¹ is an adjustment length (6.8 bp)⁶⁷. The parameters were obtained from DNA rupture experiments using magnetic tweezers (stepwise force increment, ΔF = 2–10 pN and Δt = 1 s)⁶⁸. The tension tolerance of each TGT was not experimentally measured and the value depends on loading rate which is a measure of how quickly applied force increases. Because the physiological loading rate for integrin-ligand interactions is unknown and would vary, the tension tolerance values should be considered an approximation and the relative comparison between TGTs is important. The estimation should be close to the true values when the loading rate is lower than ~10 pN/s because single-molecule optical and magnetic tweezers studies have shown that DNA unzips at 10–15 pN forces (compared to TGT-12pN, unzipping configuration) and long DNA molecules start to melt at ~60 pN (compared to TGT-54pN, shearing configuration). Recent magnetic tweezers experiment also validated the estimated tension tolerance values of TGT variants (45 and 56 pN) at loading rate of 1 pN/s²⁷.