1. Bioengineering of a single long noncoding RNA molecule that carries multiple small RNAs
- Author
-
Mei Juan Tu, Neelu Batra, Aiming Yu, Pui Yan Ho, and Hannah Petrek
- Subjects
Small interfering RNA ,Gene Expression ,Bioengineering ,Computational biology ,Biology ,Small Interfering ,medicine.disease_cause ,Applied Microbiology and Biotechnology ,Article ,Noncoding RNA ,law.invention ,03 medical and health sciences ,law ,MD Multidisciplinary ,microRNA ,Escherichia coli ,medicine ,Humans ,RNA, Small Interfering ,030304 developmental biology ,Chromatography ,Liquid ,0303 health sciences ,030306 microbiology ,Antagomirs ,General Medicine ,Non-coding RNA ,Long non-coding RNA ,Cell culture ,siRNA ,Transfer RNA ,Recombinant DNA ,RNA ,RNA, Long Noncoding ,Long Noncoding ,Lung cancer ,Chromatography, Liquid ,Biotechnology - Abstract
Noncoding RNAs (ncRNAs), including microRNAs (miRNAs), small interfering RNAs (siRNAs), and long noncoding RNAs (lncRNAs), regulate target gene expression and can be used as tools for understanding biological processes and identifying new therapeutic targets. Currently, ncRNA molecules for research and therapeutic use are limited to ncRNA mimics made by chemical synthesis. We have recently established a high-yield and cost-effective method of producing bioengineered or biologic ncRNA agents (BERAs) through bacterial fermentation, which is based on a stable tRNA/pre-miR-34a carrier (~ 180 nt) that accommodates target small RNAs. Nevertheless, it remains a challenge to heterogeneously express longer ncRNAs (e.g., > 260 nt), and it is unknown if single BERA may carry multiple small RNAs. To address this issue, we hypothesized that an additional human pre-miR-34a could be attached to the tRNA/pre-miR-34a scaffold to offer a new tRNA/pre-miR-34a/pre-miR-34a carrier (~ 296 nt) for the accommodation of multiple small RNAs. We thus designed ten different combinatorial BERAs (CO-BERAs) that include different combinations of miRNAs, siRNAs, and antagomirs. Our data showed that all target CO-BERAs were successfully expressed in Escherichia coli at high levels, greater than 40% in total bacterial RNAs. Furthermore, recombinant CO-BERAs were purified to a high degree of homogeneity by fast protein liquid chromatography methods. In addition, CO-BERAs exhibited strong anti-proliferative activities against a variety of human non-small cell lung cancer cell lines. These results support the production of long ncRNA molecules carrying different warhead small RNAs for multi-targeting which may open avenues for developing new biologic RNAs as experimental, diagnostic, and therapeutic tools.
- Published
- 2019