Protein-responsive ribozyme switches in eukaryotic cells

Abstract

Genetic devices that directly detect and respond to intracellular concentrations of proteins are impor-tant synthetic biology tools, supporting the design of biological systems that target, respond to or alter specific cellular states. Here, we develop ribozyme-based devices that respond to protein ligands in two eukaryotic hosts, yeast and mammalian cells, to reg-ulate the expression of a gene of interest. Our de-vices allow for both gene-ON and gene-OFF response upon sensing the protein ligand. As part of our de-sign process, we describe an in vitro characterization pipeline for prescreening device designs to identify promising candidates for in vivo testing. The in vivo gene-regulatory activities in the two types of eukary-otic cells correlate with in vitro cleavage activities determined at different physiologically relevant mag-nesium concentrations. Finally, localization studies with the ligand demonstrate that ribozyme switches respond to ligands present in the nucleus and/or cytoplasm, providing new insight into their mecha-nism of action. By extending the sensing capabili-ties of this important class of gene-regulatory device, our work supports the implementation of ribozyme-based devices in applications requiring the detection of protein biomarkers