title: Rational design of a small molecule-responsive intramer controlling transgene expression in mammalian cells.
abstract: Aptamers binding proteins or small molecules have been shown to be versatile and powerful building blocks for the construction of artificial genetic switches. In this study, we present a novel aptamer-based construct regulating the Tet Off system in a tetracycline-independent manner thus achieving control of transgene expression. For this purpose, a TetR protein-inhibiting aptamer was engineered for use in mammalian cells, enabling the RNA-responsive control of the tetracycline-dependent transactivator (tTA). By rationally attaching the theophylline aptamer as a sensor, the inhibitory TetR aptamer and thus tTA activity became dependent on the ligand of the sensor aptamer. Addition of the small molecule theophylline resulted in enhanced binding to the corresponding protein in vitro and in inhibition of reporter gene expression in mammalian cell lines. By using aptamers as adaptors in order to control protein activity by a predetermined small molecule, we present a simple and straightforward approach for future applications in the field of Chemical Biology. Moreover, aptamer-based control of the widely used Tet system introduces a new layer of regulation thereby facilitating the construction of more complex gene networks.
authors: ETH Zurich, Department of Biosystems Science and Bioengineering (D-BSSE), Mattenstrasse 26, CH-4058 Basel, Switzerland.
authors: David
authors: D
authors: Ausländer
authors: Markus
authors: M
authors: Wieland
authors: Simon
authors: S
authors: Ausländer
authors: Marcel
authors: M
authors: Tigges
authors: Martin
authors: M
authors: Fussenegger
mesh_terms: D000818:Animals; D052157:Aptamers, Nucleotide / Q000737:chemistry*; D001483:Base Sequence; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D005786:Gene Expression Regulation; D057809:HEK293 Cells; D006801:Humans; D008969:Molecular Sequence Data; D012097:Repressor Proteins / Q000378:metabolism; D013806:Theophylline / Q000494:pharmacology; D019076:Transgenes
chemical_list: D052157:Aptamers, Nucleotide; D012097:Repressor Proteins; C034811:tetracycline resistance-encoding transposon repressor protein; D013806:Theophylline