Nuclear hormone receptor based gene switches are ligand-regulated heterologous gene expression systems that have a wide variety of applications including gene therapy, metabolic engineering, synthetic biology, functional genomics, and animal disease models. Unfortunately, none of the existing gene switches are optimal for such applications. The major roadblock is the pleiotropic interference of the receptor-ligand combination with regulatory networks in the host organism. To address this limitation, we have used a combined directed evolution and rational design approach to engineer variants of human estrogen receptor alphaƒnligand binding domain that bind and respond to two synthetic small molecule ligands - 4,4a-dihydroxybenzil (DHB) and 2,4-di(4-hydroxyphenyl)-5-ethylthiazole (L9) - with high specificity and potency. The resulting highly specific DHB/7-S receptor and L9/L7-E receptor pairs exhibit nanomolar or subnanomolar response to their respectively matched synthetic ligand, while showing negligible or no activation in response to the natural small molecule ligand for the wild type hERa - 17b-estradiol. Furthermore, the two created receptor-ligand pairs do not show significant cross-interaction with each other. These two receptor-ligand pairs are assembled with a variety of activation/repression domains and DNA-binding domains to generate multiple gene switches for selective regulation of gene expression in mammalian cells.

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