Phosphorylation-Based miRNA High Sensor

Technology

This technology is a miRNA circuit that uses orthogonal components derived from E.coli to drive expression of a desired output. The inventors engineered E.coli histidine kinases to function either as kinases (K) or as phosphatases (P). These K and P proteins are able to positively or negatively regulate, respectively, a transcription factor (TF) that then drives expression of the desired output. MiRNA target sites specific for the high-miRNA are present on the P transcript, and the low-miRNA target sites are present on the K, TF, and output transcripts. Therefore, for the circuit to function and produce an output the high-miRNA must be present, or P will be expressed and turn off output. Additionally, the low-miRNA must be absent or K, TF, and output expression will be repressed. The inventors demonstrate that this system is robust with low basal activity and the system can achieve six fold activation of circuit output when high-miRNA is on and low-miRNA is off.

Problem Addressed

Sensing cellular biomarkers to drive a desired output allows cells to be used as miniature computational circuits. These cellular circuits can be used to generate outputs such as identifying the cell of interest with a marker, producing a desired secreted protein, or having a therapeutic effect only in cells that meet all of the circuit requirements. For example, sensing cell-type can allow for targeted killing of cancer cells that express particular markers. MicroRNA (miRNA) expression profiles differ widely across cell types, including cancer, and can therefore be used as cell-specific identifiers. Additionally, new multiplexing techniques in which one miRNA needs to be lowly expressed and another highly expressed conveys added specificity to these miRNA circuits. While low-miRNA circuits are straightforward, sensing high-miRNA expression remains a challenge. Current high-miRNA circuits rely on repressive proteins, which can introduce a time lag in the system while the repressor is being produced. Due to this design, there is often period of time in which the output is inappropriately “on” even in the wrong cell type that lacks the high-miRNA. These inventors describe a high-miRNA circuit design that eliminates this time lag by using phosphorylation driven circuitry.

Advantages

• Elimination of lag inherent in current high-miRNA systems by utilizing phosphorylation-driven circuit
• Low background activity and cross talk due to use of orthogonal proteins
• Robust circuit activation only when both activation conditions are met