Fig. 3: Validation and potential applications of an enhanced synthetic Notch receptor.

a Diagram of esNotch with an enhanced Notch core. Abbreviation: scFv, signal-chain variable fragment. TF transcription factor. NRR negative regulatory region. TMD transmembrane domain. LNR-A, LNR-B, LNR-C, HD’N, HD’C are sub-structures of NRR. S1, S2, and S3, the three proteolytic cleavage sites critical for Notch signaling^12,13. b Diagram of a synNotch similar to Fig. 1i, but with hN1RAM7 inserted between TMD and tTAA, as well as replacing mouse Notch core with human Notch core. e Diagram of a synNotch similar to b, but using CV2 as the transcription factor. Because transcription factor was changed from tTAA to CV2, pCuO driven d2EGFP was used. h Diagram of a synNotch similar to b, but using αCD19 as the scFv. k Diagram of a synNotch similar to b, but using αHer2 as the scFv. c, f, i, l For experiments as in b, e, h, k, the median d2EGFP fluorescence intensity of each co-transfection was calculated and presented as a scatter dot plot (bar: mean ± SD). Fold activation was calculated based on the mean values from synNotch cells co-cultured with sender cells with or without antigen. Groups marked by (−) were co-transfected with synNotch without the RAM sequence. d, g, j, m Similar to experiments in b–e, but without the sender cells, the median d2EGFP fluorescence intensity of each co-transfection was calculated and presented as a scatter dot plot (bar:mean ± SD). Fold change was calculated based on the mean values from 250 ng co-transfected synNotch cells without or with hN1RAM7. n Application of esNotch to generate antigen density-dependent responses. o Application of esNotches to construct complex network that could logically respond to multiple extracellular inputs.