Reprogramming chemically induced dimerization systems with genetically encoded nanobodies to control cell physiology

Background/Objectives: Protein-protein interactions (PPIs) underlie a wide range of physiological processes, and their precise control is central to synthetic biology and cellular engineering. Chemically induced proximity (CIP) systems use small molecules to regulate PPIs, enabling remote control of signaling and gene expression. Although new chemical inducers continue to emerge, repurposing established CIP chemistries to achieve new functions is a cost-effective and efficient strategy. Here, we engineered an anti-mCherry nanobody (LaM8) to create a caffeine-gated heterodimerization system and a rapamycin-triggered dissociation system. Hypothesis: We hypothesized that inserting ligand-responsive modules into permissive nanobody loops would allosterically modulate nanobody conformation and thereby enable small-molecule control of the LaM8-mCherry interaction. Methods: Loop-insertion libraries were generated by embedding either a bivalent caffeine-binding VHH module (biCOSMO) or a rapamycin-binding module (UniRapR) into LaM8. Variants were screened in mammalian cells using a cytosol-to-mitochondria translocation assay and further characterized by confocal microscopy to quantify dose-response and activation/deactivation kinetics. Lead tools were applied to regulate receptor tyrosine kinase (RTK) signaling and CRISPRa-driven gene transcription. Data/Results: From 11 caffeine-responsive variants (designated “Caffebodies”), V11 exhibited the largest caffeine-induced heterodimerization response (104%), rapid activation (t1/2 = 35.6 ± 2.3 s), and high sensitivity (EC50 = 65.8 ± 8.0 nM). This Caffebody enabled heterodimerization-based TrkA activation with low basal activity, producing a 4.8-fold increase in Ca 2+ signaling and robust ERK activation, and drove inducible gene expression with up to a 7.7-fold increase. For rapamycin-responsive variants (“Rapabodies”), V11 mediated ligand-triggered dissociation (t1/2 = 26.9 ± 2.1 s; EC50 = 172.1 ± 14.2 nM), and shutdown of CRISPRa -mediated endogenous LINE-1 expression following rapamycin addition.Conclusions: Nanobody scaffold engineering can repurpose established CIP chemistries to deliver new functions - caffeine-inducible heterodimerization and, the first rapamycin-triggered dissociation tool, enabling on-demand initiation or termination of signaling and transcriptional programs. These modules expand the chemogenetic toolkit and broaden the functional repertoire of CIP technologies to probe cell physiology. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.