Abstract
BackgroundHomeostasis within mammalian cells is achieved through complex molecular networks that can respond to changes within the cell or the environment and regulate the expression of the appropriate genes in response. The development of biological components that can respond to changes in the cellular environment and interface with endogenous molecules would enable more sophisticated genetic circuits and greatly advance our cellular engineering capabilities.ResultsHere we describe a platform that combines a ligand-responsive ribozyme switch and synthetic miRNA regulators to create an OFF genetic control device based on RNA interference (RNAi). We developed a mathematical model to highlight important design parameters in programming the quantitative performance of RNAi-based OFF control devices. By modifying the ribozyme switch integrated into the system, we demonstrated RNAi-based OFF control devices that respond to small molecule and protein ligands, including the oncogenic protein E2F1. We utilized the OFF control device platform to build a negative feedback control system that acts as a proportional controller and maintains target intracellular protein levels in response to increases in transcription rate.ConclusionsOur work describes a novel genetic device that increases the level of silencing from a miRNA in the presence of a ligand of interest, effectively creating an RNAi-based OFF control device. The OFF switch platform has the flexibility to be used to respond to both small molecule and protein ligands. Finally, the RNAi-based OFF switch can be used to implement a negative feedback control system, which maintains target protein levels around a set point level. The described RNAi-based OFF control device presents a powerful tool that will enable researchers to engineer homeostasis in mammalian cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s13036-015-0002-3) contains supplementary material, which is available to authorized users.
Highlights
Homeostasis within mammalian cells is achieved through complex molecular networks that can respond to changes within the cell or the environment and regulate the expression of the appropriate genes in response
We demonstrated RNA interference (RNAi)-based OFF control devices that respond to protein ligands by incorporating protein-responsive ribozyme switches to E2F1 and MS2
The described RNAi-based OFF control device presents a powerful tool that will enable researchers to engineer homeostasis into mammalian cells, for example, to maintain a desired phenotype even in the presence of genetic mutations or fluctuating levels of signaling molecules or cytokines
Summary
Homeostasis within mammalian cells is achieved through complex molecular networks that can respond to changes within the cell or the environment and regulate the expression of the appropriate genes in response. Molecules in the cell are organized into networks, which can respond to changes in intracellular protein or metabolite levels, as well as changes in the environment, by upregulating or downregulating the expression of any number of genes [1] These genetic circuits are essential for virtually all cellular processes including differentiation [2], replication [3], and signal transduction [4]. The pre-miRNA is subject to a final cleavage step in the cytoplasm by the enzyme Dicer, which creates a 22–25 nucleotide (nt) double stranded RNA [7] One of these RNA strands, called the guide strand, is incorporated into one of the Argonaut family of proteins to create the RNAinduced silencing complex (RISC).
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