Fig. 3: Construction of bio-orthogonal orthoSNIPR signalling systems.
From: Engineered receptors for soluble cellular communication and disease sensing
a, Left, design of a series of synthetic ligands with a range of geometries and valencies. Right, activation of an orthoLigand-responsive SNIPR driving a BFP reporter circuit in Jurkat T cells through the introduction of orthoLigands that vary in their geometry and valency (n = 6 technical replicates). b, Private and promiscuous signalling channels were created using a set of five heterodimer pairs (left). These pairs were computationally designed to have exclusive binding partners or accommodate promiscuous binding of other heterodimer components. Right, activation of five orthoSNIPRs driving a BFP reporter circuit in Jurkat T cells through the introduction of orthoLigands fused to a C6 scaffolding backbone. Ligands were added at 500 nM concentration (data are mean of n = 3 technical replicates). c, Left, schematic of a modulation strategy for orthoLigand-responsive SNIPRs through the introduction of orthoTuners generating a pseudo-C2 structure by homodimerization. Right, BFP reporter activation in Jurkat T cells after the addition of C2 orthoLigand and the orthoTuner dimerization disrupter (n = 3 technical replicates). Statistics calculated using two-way ANOVA with Å Ãdák’s multiple comparisons test. **P = 0.0077; ****P < 0.0001. d, Enhancing orthoLigands by forced lysosomal shuttling. Left, schematic of EndoTag-mediated soluble SNIPR activation. Right, activation of orthoSNIPR4 and orthoSNIPR5 in primary human T cells using recombinant orthoLigand–EndoTag 4 and 5 (respectively) 24 h after ligand exposure (n = 3 technical replicates). e, Activation of three different orthoSNIPRs in primary human T cells using their cognate and mismatched EndoTagged orthoLigands (200 nM, 24 h ligand exposure, representative of n = 3 technical replicates). f, Left, schematic of autonomous cell-to-cell communication through orthoLigand secretion. Right, activation of an orthoLigand-responsive SNIPR driving a BFP reporter circuit in Jurkat T cells through the introduction of orthoLigands secreted from sender HeLa cells (data are mean ± s.e.m. of n = 3 technical replicates). Statistics computed using one-way ANOVA with Dunnett’s multiple comparisons test.
