Molecular self-assembly under kinetic control is expected to yield nanostructures that are inaccessible through the spontaneous thermodynamic process. Moreover, time-dependent evolution, which is reminiscent of biomolecular systems, may occur under such out-of-equilibrium conditions, allowing the synthesis of supramolecular assemblies with enhanced complexities. Here we report on the capacity of a metastable porphyrin supramolecular assembly to differentiate into nanofibre and nanosheet structures. Mechanistic studies of the relationship between the molecular design and pathway complexity in the self-assembly unveiled the energy landscape that governs the unique kinetic behaviour. Based on this understanding, we could control the differentiation phenomena and achieve both one- and two-dimensional living supramolecular polymerization using an identical monomer. Furthermore, we found that the obtained nanostructures are electronically distinct, which illustrates the pathway-dependent material properties.

Most cell behaviors are the outcome of processing information from multiple signals generated upon cell stimulation. Thus, a systematic understanding of cellular systems requires methods that allow the activation of more than one specific signaling molecule or pathway within a cell. However, the construction of tools suitable for such multiplexed signal control remains challenging. In this work, we aimed to develop a platform for chemically manipulating multiple signaling molecules/pathways in living mammalian cells based on self-localizing ligand-induced protein translocation (SLIPT). SLIPT is an emerging chemogenetic tool that controls protein localization and cell signaling using synthetic self-localizing ligands (SLs). Focusing on the inner leaflet of the plasma membrane (PM), where there is a hub of intracellular signaling networks, here we present the design and engineering of two new PM-specific SLIPT systems based on an orthogonal eDHFR and SNAP-tag pair. These systems rapidly induce translocation of eDHFR- and SNAP-tag-fusion proteins from the cytoplasm to the PM specifically in a time scale of minutes upon addition of the corresponding SL. We then show that the combined use of the two systems enables chemically inducible, individual translocation of two distinct proteins in the same cell. Finally, by integrating the orthogonal SLIPT systems with fluorescent reporters, we demonstrate simultaneous multiplexed activation and fluorescence imaging of endogenous ERK and Akt activities in a single cell. Collectively, orthogonal PM-specific SLIPT systems provide a powerful new platform for multiplexed chemical signal control in living single cells, offering new opportunities for dissecting cell signaling networks and synthetic cell manipulation.

Most cell behaviors are the outcome of processing information from multiple signals generated upon cell stimulation. A systematic understanding of cellular systems requires methods that activate multiple signaling molecules or pathways in single cells. However, the construction of tools for such multiplexed signal control is challenging. Here we present orthogonal chemogenetic systems that allow control of multiple signaling pathways in living mammalian cells based on self-localizing ligand-induced protein translocation (SLIPT). Two orthogonal SLIPT systems were constructed to enable chemically inducible, individual translocation of two proteins from the cytoplasm to the inner-leaflet of the plasma membrane in the same cell. The SLIPT systems combined with fluorescent reporters achieved simultaneous multiplexed activation and monitoring of endogenous Ras/ERK and PI3K/Akt pathways in single cells. Thus, orthogonal SLIPT systems provide a powerful platform for multiplexed chemical signal control in single cells, offering new opportunities for dissecting cell signaling networks and synthetic cell manipulation.<br>