Fig. 5.

Test of dissociation of DARPin-based adapters from the virus particle. (A) Integrity of infectivity of the virus itself and the bound knob-binding modules. Black bars represent infection with Ad5luc virus alone; gray bars represent infection with virus coated with 1D3nc_SHP1 (without the HER2-binding module) at an adapter:knob ratio of 1:1. Samples were incubated in PBS at room temperature for the time points indicated before infection of BT474 cells at 100 vp per cell. RLU were determined 16 h after infection. (B) Measurements of adapter dissociation from virus. Trimeric G3_1D3nc_SHP1 adapters (gray bars) or linear trivalent (2E6)₃_G5 adapters (white bars), both containing three knob-binding DARPins and a HER2-binding DARPin (G3 or G5), were incubated with Ad5luc at a 1:1 ratio of adapter to knob at room temperature for 1.5 h in PBS before the addition of a 100-fold excess of competitor 1D3nc_SHP1 (with no HER2-binding module) (hatched bars) to prevent reassociation of dissociated adapter or no competitor (white bars) as control. Samples were incubated at room temperature for the time points indicated before infection of BT474 cells at 100 vp per cell. RLU were determined 16 h after infection. All assays were performed in duplicate; error bars show the SD.