O 87 ). A possible explanation for this increase could be the proposed bimodality of the ST-STN interaction [28]. The origin of this bimodality is believed to lie in the interaction of a single ST with a single or multiple sites on STN, where the latter is supposed to be somewhat less stable. 
Next, we performed additional experiments on (STV)biotin-DNA-Dig(AntiDig). These constructs showed an ability to sustain overstretching only in 40 of the cases, about half of what was found when using NTV and AntiDig beads. Thus, the biotin-STV linkage was significantly less stable than the biotin-NTV linkage, consistent with the significantly lower equilibrium binding constant for 1676428 biotin-NTV [23]. 
When comparing all three bead-tether-bead constructs, additional observations can be made. First, the biotin-STV linkage makes the third of these constructs weaker than the first two. Thus, the biotin-STV linkage is less stable against applied force than the tST-STN linkage. The comparison also suggests that the biotinSTV linkage is less stable than the Dig-AntiDig linkage, as the latter contributes to the second construct that is very stable. This finding may be surprising, as biotin-STV is considered to be among the most stable linkages. To address this issue we hypothesised that the way in which the linkage is establishedcould be important to stability in these experiments. Linkages can either form by incubation in bulk, during which there is a lot of time (order hour) and the molecules have many Pentagastrin web degrees of freedom. Linkages can also be formed in-situ within the tweezers apparatus by bringing the beads together, during which there is less time and fewer degrees of freedom. The former could yield more stable linkages than the latter. To test this, we performed experiments where the Dig-AntiDig connection was formed in-situ, and contrasted this with earlier results where this connection was formed by bulk incubation. In this experiment, Dig-DNA-biotin molecules were incubated with NTV-coated beads, and the Dig-AntiDig connection was formed in-situ within the tweezers. Compared to the bulk-incubated Dig connection, the results indeed showed a significant reduction in the fraction of tethers that survived overstretching: a 34 reduction in the first pull and a 25 reduction in the second pull (Figure S2). To further investigate this issue we measured the time at which the tethers broke during sustained overstretching. For incubated Dig-AntiDig linkages, 7 of tethers broke in less than a second, while for in-situ established Dig-AntiDig linkages, 67 of tethers broke within that time (Figure S3). The same unbinding time has reported for fishing Dig-AntiDig connection where DNA molecules were bound to the STV-coated beads [36]. Thus, the Dig-AntiDig connection is significantly weaker when established in-situ. The type of AntiDig antibodies used may also 15755315 affect stability. Polyclonal AntiDig antibodies are often used in single molecule PLV-2 pulling experiments [20], which could well bring significant variability in stability. The rupture force for a monoclonal AntiDig antibody was reported to be less than 20 pN for the pulling rate used in our study [37]. By incubation there may be a bias towards stronger Dig-AntiDig junctions. Importantly, in the experiments on the (STN)tST-DNA-biotin(NTV)construct (Fig. 2C), the tST-STN linkage was formed in-situ, showing that this linkage is not only stable but can also be formed rapidly. Finally, a construct consisting of (S.O 87 ). A possible explanation for this increase could be the proposed bimodality of the ST-STN interaction [28]. The origin of this bimodality is believed to lie in the interaction of a single ST with a single or multiple sites on STN, where the latter is supposed to be somewhat less stable. Next, we performed additional experiments on (STV)biotin-DNA-Dig(AntiDig). These constructs showed an ability to sustain overstretching only in 40 of the cases, about half of what was found when using NTV and AntiDig beads. Thus, the biotin-STV linkage was significantly less stable than the biotin-NTV linkage, consistent with the significantly lower equilibrium binding constant for 1676428 biotin-NTV [23]. When comparing all three bead-tether-bead constructs, additional observations can be made. First, the biotin-STV linkage makes the third of these constructs weaker than the first two. Thus, the biotin-STV linkage is less stable against applied force than the tST-STN linkage. The comparison also suggests that the biotinSTV linkage is less stable than the Dig-AntiDig linkage, as the latter contributes to the second construct that is very stable. This finding may be surprising, as biotin-STV is considered to be among the most stable linkages. To address this issue we hypothesised that the way in which the linkage is establishedcould be important to stability in these experiments. Linkages can either form by incubation in bulk, during which there is a lot of time (order hour) and the molecules have many degrees of freedom. Linkages can also be formed in-situ within the tweezers apparatus by bringing the beads together, during which there is less time and fewer degrees of freedom. The former could yield more stable linkages than the latter. To test this, we performed experiments where the Dig-AntiDig connection was formed in-situ, and contrasted this with earlier results where this connection was formed by bulk incubation. In this experiment, Dig-DNA-biotin molecules were incubated with NTV-coated beads, and the Dig-AntiDig connection was formed in-situ within the tweezers. Compared to the bulk-incubated Dig connection, the results indeed showed a significant reduction in the fraction of tethers that survived overstretching: a 34 reduction in the first pull and a 25 reduction in the second pull (Figure S2). To further investigate this issue we measured the time at which the tethers broke during sustained overstretching. For incubated Dig-AntiDig linkages, 7 of tethers broke in less than a second, while for in-situ established Dig-AntiDig linkages, 67 of tethers broke within that time (Figure S3). The same unbinding time has reported for fishing Dig-AntiDig connection where DNA molecules were bound to the STV-coated beads [36]. Thus, the Dig-AntiDig connection is significantly weaker when established in-situ. The type of AntiDig antibodies used may also 15755315 affect stability. Polyclonal AntiDig antibodies are often used in single molecule pulling experiments [20], which could well bring significant variability in stability. The rupture force for a monoclonal AntiDig antibody was reported to be less than 20 pN for the pulling rate used in our study [37]. By incubation there may be a bias towards stronger Dig-AntiDig junctions. Importantly, in the experiments on the (STN)tST-DNA-biotin(NTV)construct (Fig. 2C), the tST-STN linkage was formed in-situ, showing that this linkage is not only stable but can also be formed rapidly. Finally, a construct consisting of (S.