Elucidation and refinement of synthetic receptor mechanisms

Abstract

Synthetic receptors are powerful tools for engineering mammalian cell-based devices. These biosensors confer unique capabilities for detecting environmental ligands and transducing signals to control downstream gene expression events such as therapeutic protein production. For many applications, it would be useful to understand how receptor design choices impart desirable performance metrics and trade-offs. Towards this goal, we employed the existing modular extracellular sensor architecture (MESA) and systematically characterized biosensors with previously unexamined protein domain choices. A key finding that might extend to other receptor systems is that choice of transmembrane domain (TMD) is highly consequential. To provide mechanistic insights, we adopted and employed a FRET-based assay to elucidate how TMDs affect receptor complex formation and connected these observations to functional performance. To build further insight into these phenomena, we developed a library of new MESA receptors that sense an expanded set of ligands. Based upon these explorations, we conclude that TMDs affect signaling primarily by modulating intracellular domain geometry, and we apply this understanding to rationally tune receptors. Finally, to guide the design of future receptors, we propose general principles for linking design choices to biophysical mechanisms and performance characteristics.

Model-guided design of mammalian genetic programs

“…For Rapa-MESA-ZF6a (ZF6a was also selected for its potency), the ligand-inducible fold difference in signal was ~200x (Fig. 4B), which is several fold higher than was observed for recently reported receptors based on tTA (46), and also higher than the fold difference observed for a high-performing MESA that employs a distinct mechanism (47). Thus, Rapa-MESA-ZF6a is the highest performing MESA reported to date.…”

“…In this mechanism, ligand-mediated dimerization of two transmembrane proteins called the target chain (TC) and protease chain (PC) promotes PC-mediated proteolytic trans-cleavage of a TC-bound TF. We explored several strategies for building COMET-compatible MESA based on a recently reported improved MESA design (46) and the parts developed in the current study ( Fig. S4B-G).…”

Protease-controlled secretion and display of intercellular signals

“…Backbones were linearized via restriction digestion, and inserts were generated using PCR, or purchased from Twist Biosciences. MESA-rapalog receptor source plasmids were a generous gift from Joshua Leonard 26 . The plasmid containing the voltage indicator, ASAP3 was a generous gift from Michael Lin 54 .…”

“…As a proof of principle, we used the well-established MESA receptor (membranelocalized split TEVP reconstituted by rapalog) 25,26,73 as an input to activate RELEASE via the intermediate protease circuit optimized above (Fig. 4c).…”

“…For analysis, we selected and compared cells with the highest expression of the cotransfection marker, which was typically mCherry. This was done to have the largest separation between basal reporter autofluorescence from cellular autofluorescence, as previously described 7,26 . For experiments using the Kir2.1 potassium channel, cells were either cotransfected with the voltage indicator ASAP3 54 or incubated with the Oxonol chemical dye, DiSBAC2(3) as previously described 55 .…”