Linker-Length Landscape Mapping Enables Coupling of Diverse Synthetic Chemically Induced Dimerization Systems to Molecular Readouts

Programmable molecular biology increasingly requires strategies for converting engineered recognition or proximity modules into measurable outputs, particularly within transcriptional regulation, RNA imaging, and CRISPR-associated systems. Synthetic chemically induced dimerization (CID) systems provide a class of programmable recognition modules for such applications, yet generalized strategies for coupling structurally diverse CIDs to functional readouts remain limited. Here, we introduce a CID-to-output conversion strategy based on engineering of the linker-mediated coupling interface. Using single-fluorescent-protein sensors as an experimentally tractable optical model readout, we systematically varied paired N- and C-terminal linkers flanking circularly permuted green fluorescent protein (cpGFP) to map coupling landscapes across synthetic CID systems derived from combinatorial selection and computational protein design. The results revealed strong non-additive interactions across paired linkers and suggest that linker length is a first-order determinant of CID-to-output coupling. Across nanobody-, monobody-, and de novo-designed CID architectures, this framework yielded functional sensors with dynamic ranges up to 1270% and robust responses in mammalian cells. Together, this work demonstrates that effective CID-to-output conversion can be achieved by empirically mapping the linker-mediated coupling interface, providing a practical route for adapting synthetic CID to diverse programmable molecular readouts and nucleic-acid-associated synthetic biology systems.