Researcher(s)
- Jacob Nicosia, Chemical Engineering, University of Delaware
Faculty Mentor(s)
- Wilfred Chen, Chemical Engineering, University of Delaware
- Mark Blenner, Chemical Engineering, University of Delaware
Abstract
Proteins are the engine of the cell, facilitating all of the reactions that take place and driving cellular function. Tools to control protein residence time within the cell could have broad applications as therapeutics, or aid in the study of intracellular proteins.
This project investigates the use of metabolite-responsive aptamers to conditionally degrade proteins in response to a signal molecule, building on a recently published system using a destabilized MS2 coat protein (MCP). MCP is an RNA-binding protein, and this version is engineered to be unstable by the attachment of a C-terminal degron tag, an amino acid sequence marking proteins for degradation. The unstable MCP protein is fused to a fluorescent protein to allow quantification via flow cytometry and microscopy. The degron tag is strategically placed adjacent to the RNA binding site, such that when the RNA binds, it blocks the cell from recognizing the degron tag and prevents the protein from being degraded. The specific RNA that binds to MCP is called the MS2 aptamer; in the original system, this aptamer is always in the MCP-binding form, and thus it always binds to MCP when it is present. The system we are proposing uses a metabolite responsive MS2 aptamer, a unique version of this aptamer that only folds into the MCP-binding form when it binds a target molecule. Using these metabolite-responsive aptamers would allow the degradation to be controlled by metabolite concentration rather than the presence of RNA alone; the aptamer will only bind and block degradation if it folds into the correct shape, which would be when its target molecule is bound. While the system at first would be linked with the previously mentioned fluorescent proteins to allow quantification, it could serve as a platform to build therapeutic tools by swapping out the input (the target molecule bound by aptamer) and the output (what protein is targeted for degradation).