A Signal Inverter Module for RNA Cleavers: Detecting and Evaluating miRNA, Ribozymes, and Ribonucleases

Technology

This technology is an all-in-one system to sense high levels of a miRNA that eliminates the time lag inherent in most high-miRNA circuitry. To accomplish this, the inventors designed a transcript that has the desired output coding sequence followed by a stabilizer, a miRNA target or ribonuclease cleavage sequence, and finally a degradation signal. Under conditions where the miRNA or ribozyme is not present, the transcript is degraded due to the degradation signal. When the miRNA or ribonuclease is present in the cell, the transcript is cleaved at the miRNA or ribonuclease recognition site, therefore removing the degradation signal from the transcript and allowing expression of the output protein. This tool provides a novel way of designing a miRNA high sensor for miRNA circuits, and it can additionally be used in research applications to test ribozyme and ribonuclease activity.

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” for a period of time even in cells without high levels of the miRNA. These inventors describe a high-miRNA sensing circuit design that eliminates this time lag by inverting a traditional miRNA system to yield a gain-of-signal output.

Advantages

• Elimination of lag time inherent in current miRNA high sensors
• Gain of signal assay for testing of miRNA, ribozyme, and ribonuclease activity