RNA origami enables applications in synthetic biology

Developing tools for precise control of biological processes has been one of the main pillars of the now mature field of synthetic biology. These scientific tools borrow principles from a wide range of research areas that, when combined, enable unique applications that are potentially transformative for modern society.

Translating modern RNA nanotechnology innovations into the biological context has enormous potential due to its compatibility with folding and expression in cells, but it also poses unique challenges such as tight performance conditions and inherent instability of RNA molecules.

However, a recent approach to structural RNA design developed in Andersen’s lab called “RNA origami” seeks to address this. This approach seeks to generate complex man-made RNA-based devices that are stable in cells, interact with other biomolecules, including other RNA and proteins, and enable unique applications, particularly in the context of gene regulation.

Demonstrated by two different approaches recently published in: Nucleic Acid ResearchRNA origami is presented as an advanced RNA design platform that, when applied in the cellular context, generates unique molecules for synthetic biology-based regulation.

RNA sponges regulate the production of enzymes in bacteria

In the first approach, the RNA origami was used to achieve precise control of the protein production levels when expressed in bacteria. Self-inhibiting protein expression cassettes were made by installing a strong binding site for the expressed protein in its own gene. Thereafter, RNA origami decorated with the same protein binding sites was overexpressed.

In this way, the RNA origami serves as a protein sponge that sequesters proteins in the cell and allows expression of the self-inhibited protein. This general concept was found to allow for the regulation of several proteins simultaneously and enable enzymatic pathways for improved product yields.

CRISPR-based regulators for chemical yeast plants

In the second approach, RNA origami was combined with CRISPR, one of the most popular modern molecular biology techniques, to regulate gene expression in yeast. The RNA origamis were integrated into the small RNAs that direct CRISPR-Cas9 to target specific sequences in the DNA genome.

The RNA origami scaffolds were decorated with protein binding sites that could recruit transcription factors. By targeting the RNA scaffolds to promoter regions, the transcription factors activated gene expression. It was shown that the expression strength can be tuned by the orientation of the scaffold and the amount of recruited transcription factors. Finally, it was shown that multi-enzyme pathways can be controlled for the production of high yields of the cancer-fighting drug violacein.