The impact of 4-thiouridine posttranscriptional modification on the oncosuppressive activity of 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 (m 5 U) 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 (s 4 U) modification at position 8 (s 4 U8) of the human tRNA segment. To re-innovate our BioRNA technology and generate BioRNA/let-7e-5p molecules free of s 4 U 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 s 4 U 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 s 4 U modification. Surprisingly, comprehensive comparative studies showed minimal effects of the distal s 4 U 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.