Extended Data Fig. 2: Comparison of binding affinities from freshly made and 30-day-old samples, and mitochondria immunostainings in control U2OS cells.

a, Little decrease in binding observed for designs RPB_PLP1_R6 and RPB_PEW1_R6 30-day-old in 4 °C. Bio-layer interferometry characterization of binding of designed proteins to the corresponding peptide targets. Twofold serial dilutions were tested for each binder, and the full tested concentration is labelled. The biotinylated target peptides were loaded onto the streptavidin (SA) biosensors, and incubated with designed binders in solution to measure association and dissociation. b, Mitochondria immunostainings in control U2OS cells. Wild-type U2OS cells were spread onto fibronectin coverslips as in Fig. 3, then fixed and processed for immunofluorescence using TOM20 antibodies as a marker of mitochondria. Note that mitochondria appearance in these control cells is similar to that observed upon overexpression of designed binders fused to mitochondria-targeting sequences (Fig. 3). suggesting that these constructs do not affect mitochondria shape. Scale bar, 10 µm.