Bioengineering of a single long noncoding RNA molecule that carries multiple small RNAs
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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.
KeywordsNoncoding RNA microRNA siRNA Bioengineering Lung cancer
This study was supported by the National Cancer Institute (grant no. R01CA225958) and National Institute of General Medical Sciences (R01GM113888), National Institutes of Health. The authors also appreciate the access to the Molecular Pharmacology Shared Resources funded by the UC Davis Comprehensive Cancer Center Support Grant (CCSG) awarded by the National Cancer Institute (P30CA093373).
Compliance with ethical standards
The authors confirm that the article does not contain any studies with human participants or animals.
Conflict of interest
The authors are named inventors of patent applications related to RNA bioengineering technology and therapeutics that are owned by the UC Davis, and Dr. Yu is a founder of AimRNA, Inc., which has an agreement to license the intellectual property. All other authors declare that they have no conflict of interest.
- Bar-Peled L, Kemper EK, Suciu RM, Vinogradova EV, Backus KM, Horning BD, Paul TA, Ichu TA, Svensson RU, Olucha J, Chang MW, Kok BP, Zhu Z, Ihle NT, Dix MM, Jiang P, Hayward MM, Saez E, Shaw RJ, Cravatt BF (2017) Chemical proteomics identifies druggable vulnerabilities in a genetically defined cancer. Cell 171(3):696–709 e23CrossRefGoogle Scholar
- Jian C, Tu MJ, Ho PY, Duan Z, Zhang Q, Qiu JX, DeVere White RW, Wun T, Lara PN, Lam KS, Yu AX, Yu AM (2017) Co-targeting of DNA, RNA, and protein molecules provides optimal outcomes for treating osteosarcoma and pulmonary metastasis in spontaneous and experimental metastasis mouse models. Oncotarget 8(19):30742–30755CrossRefGoogle Scholar
- Li MM, Addepalli B, Tu MJ, Chen QX, Wang WP, Limbach PA, LaSalle JM, Zeng S, Huang M, Yu AM (2015) Chimeric microRNA-1291 biosynthesized efficiently in Escherichia coli is effective to reduce target gene expression in human carcinoma cells and improve chemosensitivity. Drug Metab Dispos 43(7):1129–1136CrossRefGoogle Scholar
- Li X, Tian Y, Tu MJ, Ho PY, Batra N, Yu AM (2019) Bioengineered miR-27b-3p and miR-328-3p modulate drug metabolism and disposition via the regulation of target ADME gene expression. Acta Pharm Sin B. https://doi.org/10.1016/j.apsb.2018.12.002
- Pitulle C, Hedenstierna KO, Fox GE (1995) A novel approach for monitoring genetically engineered microorganisms by using artificial, stable RNAs. Appl Environ Microbiol 61(10):3661–3666Google Scholar
- Rosano GL, Ceccarelli EA (2014) Recombinant protein expression in Escherichia coli: advances and challenges. Front Microbiol 5:172Google Scholar
- Wang WP, Ho PY, Chen QX, Addepalli B, Limbach PA, Li MM, Wu WJ, Jilek JL, Qiu JX, Zhang HJ, Li T, Wun T, White RD, Lam KS, Yu AM (2015) Bioengineering novel chimeric microRNA-34a for prodrug cancer therapy: high-yield expression and purification, and structural and functional characterization. J Pharmacol Exp Ther 354(2):131–141CrossRefGoogle Scholar