The invention addresses the need for methods to reproducibly and predictably manipulate gene expression in a desired organism or cell-free biological system. The amount of target protein is tightly controlled by the addition of a regulatory compound or an endogenous molecule.
Current inducible gene expression systems focus on the regulation of DNA transcription by allowing users to control the interactions between RNA polymerases and their DNA template. Inducible translation systems include ligand-binding aptarners (riboswitches) that are capable of affecting local mRNA structure in response to a ligand. Alternative systems allow users to control target mRNA or protein stability by the use of small-molecule-responsive autoendonucleases or by the use of degradation enhancer domains, respectively. However, these systems are highly specific in the factors to which they respond, and cannot be infinitely expanded into multiple, orthogonal inducible systems.
The present invention comprehends an extensible and modular system which allows users to combine a given repressor protein (RP) aptamer pair with a given gene of interest, an improvement over riboswitches which are highly context-dependent and have only been developed for a limited set of ligands. In addition, this invention is expandable, since a given repressor protein can be fused with a second protein conveying an additional activity, binding specificity, or subcellular localization.
This invention could be applied to regulate the activity of RNA aptamer-based therapeutics and/or to develop medical diagnostic devices. Alternatively, it has potential applications in environmental safety monitoring or as biosensor for exogenous or endogenous compounds.
This invention consists of a system comprising: (1) A repressor protein (RP) that is either produced within a cell or added to a cell-free biological system; (2) A regulatory RNA sequence that when added in single or multiple copy to the 5' or 3' untranslated region (UTR) of a gene encoding the target protein, is capable of forming an RNA structure (aptamer) that binds the RP (1) with high affinity in the context of a cellular environment or a cell-free system; and (3) a regulatory molecule capable of interacting with the RP and altering its interaction with the aptamer (2).
- Reproducible and predictable gene expression manipulation for desire amounts of protein target
- Expandable system, allows for the combination of a given repressor protein-aptamer pair to any given gene of interest
- Additional activity derived from fusing the repressor proteins to a second protein for binding specificity, or subcellular localization control