Bioengineering of a single long noncoding RNA molecule that carries multiple small RNAs

Area of Science:

• Biotechnology
• Molecular Biology
• RNA Therapeutics

Background:

• Noncoding RNAs (ncRNAs) regulate gene expression and are valuable research and therapeutic tools.
• Current ncRNA production relies on chemical synthesis, limiting availability and increasing costs.
• Bioengineered or biologic ncRNA agents (BERAs) produced via bacterial fermentation offer a high-yield, cost-effective alternative.

Purpose of the Study:

• To develop a method for producing longer ncRNAs (>260 nt) capable of carrying multiple small RNAs.
• To engineer a novel tRNA/pre-miR-34a/pre-miR-34a carrier for accommodating multiple small RNAs.
• To create combinatorial BERAs (CO-BERAs) for multi-targeting strategies.

Main Methods:

• Designed ten combinatorial BERAs (CO-BERAs) with various combinations of miRNAs, siRNAs, and antagomirs.
• Expressed CO-BERAs in Escherichia coli, achieving high yields (>40% of total bacterial RNAs).
• Purified recombinant CO-BERAs using fast protein liquid chromatography.

Main Results:

• All designed CO-BERAs were successfully expressed at high levels in E. coli.
• Purified CO-BERAs demonstrated high homogeneity.
• CO-BERAs exhibited significant anti-proliferative activity against non-small cell lung cancer cell lines.

Conclusions:

• Bacterial fermentation can produce long ncRNA molecules carrying multiple small RNAs for multi-targeting.
• This approach facilitates the development of novel biologic RNAs for experimental, diagnostic, and therapeutic applications.
• CO-BERAs represent a promising platform for advancing RNA-based research and therapies.