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Abstract

Proximity-dependent protein interactions are crucial for many cellular processes, which makes methods to precisely control protein localization highly desirable. Chemical inducers of proximity (CIPs) are a tool that enables such manipulation by bringing specific protein domains together that can be fused to proteins of interest (POIs). This work focusses on the development of different strategies for precise control of protein localization in live cells and in vivo derived from plant hormone-based CIPs. To introduce a further control over the chemically induced protein proximity, I developed Mandi-Dopa-C6-Indole, an antagonist for the plant hormone-based CIP mandipropamid (Mandi), that is able to reverse protein proximity previously induced by Mandi in live mammalian cells. Moreover, I demonstrate that pMandi, a photocaged derivative of Mandi, can be used to induce protein proximity in live mammalian cells and in live medaka embryos upon light irradiation as external stimulus. Although pMandi enables precise temporal control over the induction of protein proximity, spatial control is limited by the high permeability of Mandi. For the design of a photocaged CIP that enables also precise spatial control over the induction of protein proximity with single-cell resolution, I repurposed the abscisic acid (ABA) agonist opabactin (OP) from plant research as CIP. The carboxylic acid moiety of OP renders the molecule less cell permeable and enables intracellular trapping. When the carboxylic acid moiety is protected as acetoxymethyl (AM) ester, the addition of this compound induces protein proximity in live mammalian cells and in live medaka embryos at low working concentrations comparable to Mandi. The photocaged derivative of OP, pOP, allows inducing protein proximity upon irradiation with light in single live mammalian cells and enables the manipulation of individual cells in the same sample. To extend the CIP system based on Mandi from dimerization to trimerization, I split one of the interacting proteins into two fragments and demonstrated that the fragments can reconstitute and recruit the second interacting protein in the presence of the CIP in live mammalian cells when spatial proximity of the split fragments is given. I further successfully applied this split system to construct a logic gate in combination with the CIP rapamycin.