Expression and Purification of tRNA/ pre-miRNA-Based Recombinant Noncoding RNAs

Area of Science:

• Biotechnology and Molecular Biology
• RNA Therapeutics

Background:

• Current RNA therapeutics often rely on chemoengineered RNA mimics, which may not fully replicate natural RNA properties.
• Bioengineered RNA molecules produced in living cells offer potential for improved structure, function, and safety profiles.
• Novel technologies are emerging for recombinant RNA production, necessitating robust manufacturing platforms.

Purpose of the Study:

• To develop a reproducible fermentation method for producing bioengineered RNA agents (BERAs).
• To demonstrate the utility of BERAs for carrying various small RNA payloads, including miRNAs, siRNAs, and aptamers.
• To establish a scalable and efficient purification process for high-purity BERAs.

Main Methods:

• Development of an optimal hybrid tRNA/pre-miRNA carrier for BERA construction.
• Fermentation-based production of BERAs in a bacterial system.
• Purification of target BERAs using fast protein liquid chromatography (FPLC).

Main Results:

• Achieved reproducible fermentation production of BERAs with high-level expression (>30% of total bacterial RNAs).
• Successfully purified BERAs carrying various small RNAs (miRNAs, siRNAs, aptamers) to high homogeneity (>97%).
• Demonstrated large-scale production capability, yielding multiple to tens of milligrams of ready-to-use BERAs per liter of bacterial culture.

Conclusions:

• The described fermentation approach provides a robust and scalable method for producing bioengineered RNA agents (BERAs).
• BERAs offer a versatile platform for delivering diverse small RNA payloads with potential therapeutic applications.
• This technology enables consistent, high-yield production of purified BERAs, facilitating their advancement into preclinical and clinical studies.