A miRNA-Based NOT Gate As a Building Block of More Sophisticated Circuits in Mammalian Cells

Scaling up genetic circuits in mammalian cells can lead to a new class of therapies. However, large DNA footprints limit in the choice of safer gene delivery platform like the AAV viral vector, which has a small package capability at about 4.7kb. On the other hand, the use of exogenous proteins can limit the applications in vivo because they can trigger dangerous immune reactions.

Here, we show a miRNA-based NOT gate that has a small DNA footprint and that does not use transcriptional regulators or, more in general, exogenous proteins. This can be the building block of more sophisticated information processing in mammalian cells.

Active miRNAs recognize their target RNA through Watson and Crick base-pairing, which in turn provides a large design space. Additionally input and output signals are biomolecules of the same type. Because of that, miRNA-based NOT gates might be easily combined to make more complex logic circuits.

This NOT gate was implemented with just one transcriptional unit by modifying a ncRNA that naturally shuttles between the nucleus and the cytoplasm. This design successfully showed a NOT-gate behavior in HEK293FT after transiently transfecting the DNA encoding the gate and the input as well.

We are currently investigating the use of the miRNA-based NOT gate for more complex information processing. We are confident that it will push the boundary of what currently can be computed in mammalian cells.