Figure 1

Design of rigid DARPin-DARPin fusions. (a) Rigid fusions of two or more DARPins can be generated by joining the C-terminal helix of one DARPin to the N-terminal helix of a second DARPin. The length of the shared helix should optimally be less than the sum of the lengths of the individual helices, ensuring that the helix is embedded in at least one of the two domains along its entire length. The overall geometry of the construct depends on the length of the shared helix. Since the C-and the N-terminal helix of a DARPin run roughly antiparallel to each other, this results in the two paratopes facing in opposite directions, minimizing the probability of target proteins bound to the two paratopes clashing with each other. Capping repeats are shown in white, the terminal helices giving rise to the shared helix in pink, the internal repeats carrying the DARPin paratope in yellow. Residues randomized in the DARPin library, giving rise to the paratope in selected binders, are highlighted in orange. (b) Genetic organization of the constructs. The binding specificity of each DARPin is encoded in the sequence of its internal repeats (I1 to I3, etc.). The number of internal repeats can vary, three being most common. A non-randomized spacer repeat can be inserted between the randomized repeats and the connector module to adjust the spacing between the two paratopes and to avoid loss of affinity if the paratope of a selected binder extends onto the capping repeat. To facilitate the exchange of DARPin specificities, unique restriction sites were introduced between internal repeats and connector modules. (c) Sequences of the connector modules joining two DARPins. Residues retained from the original capping repeats (taken from a consensus DARPin with stabilized C-cap, mut5, PDB ID: 2XEE²²) are shown in white and pink, residues changed by Rosetta fixbb ¹⁷ sequence optimization are highlighted in red.