Impact of 4-Thiouridine Modification on Bioengineered Let-7e-5p in NSCLC Cells

Current microRNA (miRNA) research and therapeutic development primarily rely on miRNA mimics chemically synthesized in vitro. While effective, miRNA mimics may not fully recapitulate the properties of natural miRNA agents synthesized in vivo. Our lab has developed a novel tRNA-fused pre-miRNA carrier-based RNA bioengineering platform technology, enabling consistent, high-yield, and large-scale production of biologic miRNAs (BioRNAs) through in vivo bacterial fermentation, which have shown strong potential in experimental therapeutics for non-small cell lung cancer (NSCLC). Among the tumor suppressive let-7-5p isoforms, BioRNA/let-7e-5p was identified as the most effective to suppress human NSCLC cell viability. Further, while we observed species-conserved posttranscriptional modifications, such as dihydrouridine (D) and 2′-O-methylguanosine (Gm) in the D-loop as well as 5-methyluridine (m5U) and pseudouridine (Y) in the T-loop, liquid chromatography tandem mass spectrometry (LC-MS/MS) and nanopore-based direct RNA sequencing studies revealed the presence of a bacteria-specific 4-thiouridine (s4U) modification at position 8 (s4U8) of the human tRNA segment. To re-innovate our BioRNA technology and generate BioRNA/let-7e-5p molecules free of s4U modification, we employed two strategies: (1) change of U8 through deletion or substitutions, and (2) expression of U8 BioRNAs in an E. coli strain deficient in s4U synthesis. Both approaches proved successful, allowing us to produce milligrams of pure, ready-to-use BioRNA/let-7e-5p molecules per 200 mL bacterial culture, and LC-MS/MS and nanopore sequencing analyses confirmed the escape from s4U modification. Surprisingly, comprehensive comparative studies showed minimal effects of the distal s4U modification on the release of let-7e-5p from BioRNAs and consequently, downregulation of multiple targeted oncogenes in NSCLC cells. These findings will guide the development of BioRNA/let-7e-5p as potential therapeutics for NSCLC treatment. Citation Format: Katherine K. Wang, Mei-Juan Tu, Yufan Zhou, Ziyuan Wang, Patrick A. Limbach, Hongxu Ding, Ai-Ming Yu. The impact of 4-thiouridine posttranscriptional modification on the oncosuppressive activity of bioengineered let-7e-5p in NSCLC cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 2063.