Molecular Engineering of Functional SiRNA Agents.

Synthetic biology constitutes a scientific domain focused on intentional redesign of organisms to confer novel functionalities or create new products through strategic engineering of their genetic makeup. Leveraging the inherent capabilities of nature, one may address challenges across diverse sectors including medicine. Inspired by this concept, we have developed an innovative bioengineering platform, enabling high-yield and large-scale production of biological small interfering RNA (BioRNA/siRNA) agents via bacterial fermentation. Herein, we show that with the use of a new tRNA fused pre-miRNA carrier, we can produce various forms of BioRNA/siRNA agents within living host cells. We report a high-level overexpression of nine target BioRNA/siRNA molecules at 100% success rate, yielding 3-10 mg of BioRNA/siRNA per 0.25 L of bacterial culture with high purity (>98%) and low endotoxin (<5 EU/μg RNA). Furthermore, we demonstrate that three representative BioRNA/siRNAs against GFP, BCL2, and PD-L1 are biologically active and can specifically and efficiently silence their respective targets with the potential to effectively produce downstream antiproliferation effects by PD-L1-siRNA. With these promising results, we aim to advance the field of synthetic biology by offering a novel platform to bioengineer functional siRNA agents for research and drug development.