Advertisement

Bioengineering of a single long noncoding RNA molecule that carries multiple small RNAs

  • Hannah Petrek
  • Neelu Batra
  • Pui Yan Ho
  • Mei-Juan Tu
  • Ai-Ming YuEmail author
Biotechnological products and process engineering
  • 46 Downloads

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.

Keywords

Noncoding RNA microRNA siRNA Bioengineering Lung cancer 

Notes

Funding information

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

Ethical statement

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.

References

  1. Alegre F, Ormonde AR, Snider KM, Woolard K, Yu AM, Wittenburg LA (2018) A genetically engineered microRNA-34a prodrug demonstrates anti-tumor activity in a canine model of osteosarcoma. PLoS One 13(12):e0209941CrossRefGoogle Scholar
  2. Ambros V (2004) The functions of animal microRNAs. Nature 431(7006):350–355CrossRefGoogle Scholar
  3. Asangani IA, Rasheed SA, Nikolova DA, Leupold JH, Colburn NH, Post S, Allgayer H (2008) MicroRNA-21 (miR-21) post-transcriptionally downregulates tumor suppressor Pdcd4 and stimulates invasion, intravasation and metastasis in colorectal cancer. Oncogene 27(15):2128–2136CrossRefGoogle Scholar
  4. 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
  5. Bramsen JB, Kjems J (2012) Development of therapeutic-grade small interfering RNAs by chemical engineering. Front Genet 3:154CrossRefGoogle Scholar
  6. Chen QX, Wang WP, Zeng S, Urayama S, Yu AM (2015) A general approach to high-yield biosynthesis of chimeric RNAs bearing various types of functional small RNAs for broad applications. Nucleic Acids Res 43(7):3857–3869CrossRefGoogle Scholar
  7. Esteller M (2011) Non-coding RNAs in human disease. Nat Rev Genet 12(12):861–874CrossRefGoogle Scholar
  8. Gaudin C, Nonin-Lecomte S, Tisné C, Corvaisier S, Bordeau V, Dardel F, Felden B (2003) The tRNA-like domains of E. coli and A. aeolicus transfer–messenger RNA: structural and functional studies. J Mol Biol 331(2):457–471CrossRefGoogle Scholar
  9. Hatziapostolou M, Polytarchou C, Aggelidou E, Drakaki A, Poultsides GA, Jaeger SA, Ogata H, Karin M, Struhl K, Hadzopoulou-Cladaras M, Iliopoulos D (2011) An HNF4α-miRNA inflammatory feedback circuit regulates hepatocellular oncogenesis. Cell 147(6):1233–1247CrossRefGoogle Scholar
  10. Hermeking H (2010) The miR-34 family in cancer and apoptosis. Cell Death Differ 17(2):193–199CrossRefGoogle Scholar
  11. Ho PY, Yu AM (2016) Bioengineering of noncoding RNAs for research agents and therapeutics. Wiley Interdiscip Rev RNA 7(2):186–197CrossRefGoogle Scholar
  12. Ho PY, Duan Z, Batra N, Jilek JL, Tu MJ, Qiu JX, Hu Z, Wun T, Lara PN, DeVere White RW, Chen HW, Yu AM (2018) Bioengineered noncoding RNAs selectively change cellular miRNome profiles for cancer therapy. J Pharmacol Exp Ther 365(3):494–506CrossRefGoogle Scholar
  13. Hutchinson CR (1998) Combinatorial biosynthesis for new drug discovery. Curr Opin Microbiol 1(3):319–329CrossRefGoogle Scholar
  14. 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
  15. Jilek JL, Zhang QY, Tu MJ, Ho PY, Duan Z, Qiu JX, Yu AM (2019) Bioengineered let-7c inhibits orthotopic hepatocellular carcinoma and improves overall survival with minimal immunogenicity. Mol Ther Nucleic Acids 14:498–508CrossRefGoogle Scholar
  16. Johnson SM, Grosshans H, Shingara J, Byrom M, Jarvis R, Cheng A, Labourier E, Reinert KL, Brown D, Slack FJ (2005) RAS is regulated by the let-7 microRNA family. Cell 120(5):635–647CrossRefGoogle Scholar
  17. Khvorova A, Watts JK (2017) The chemical evolution of oligonucleotide therapies of clinical utility. Nat Biotechnol 35(3):238–248CrossRefGoogle Scholar
  18. Knight V, Sanglier JJ, DiTullio D, Braccili S, Bonner P, Waters J, Hughes D, Zhang L (2003) Diversifying microbial natural products for drug discovery. Appl Microbiol Biotechnol 62(5–6):446–458CrossRefGoogle Scholar
  19. Leader B, Baca QJ, Golan DE (2008) Protein therapeutics: a summary and pharmacological classification. Nat Rev Drug Discov 7(1):21–39CrossRefGoogle Scholar
  20. Levin AA (2019) Treating disease at the RNA level with oligonucleotides. N Engl J Med 380(1):57–70CrossRefGoogle Scholar
  21. Li MM, Wang WP, Wu WJ, Huang M, Yu AM (2014) Rapid production of novel pre-microRNA agent hsa-mir-27b in Escherichia coli using recombinant RNA technology for functional studies in mammalian cells. Drug Metab Dispos 42(11):1791–1795CrossRefGoogle Scholar
  22. 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
  23. Li PC, Tu MJ, Ho PY, Jilek JL, Duan Z, Zhang QY, Yu AX, Yu AM (2018) Bioengineered NRF2-siRNA is effective to interfere with NRF2 pathways and improve chemosensitivity of human cancer cells. Drug Metab Dispos 46(1):2–10CrossRefGoogle Scholar
  24. 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
  25. Lin PY, Yu SL, Yang PC (2010) MicroRNA in lung cancer. Br J Cancer 103(8):1144–1148CrossRefGoogle Scholar
  26. Liu YP, Berkhout B (2011) miRNA cassettes in viral vectors: problems and solutions. Biochim Biophys Acta 1809(11–12):732–745CrossRefGoogle Scholar
  27. Liu Y, Stepanov VG, Strych U, Willson RC, Jackson GW, Fox GE (2010) DNAzyme-mediated recovery of small recombinant RNAs from a 5S rRNA-derived chimera expressed in Escherichia coli. BMC Biotechnol 10:85CrossRefGoogle Scholar
  28. Meng F, Henson R, Wehbe-Janek H, Ghoshal K, Jacob ST, Patel T (2007) MicroRNA-21 regulates expression of the PTEN tumor suppressor gene in human hepatocellular cancer. Gastroenterology 133(2):647–658CrossRefGoogle Scholar
  29. Nelissen FHT, Leunissen EHP, van de Laar L, Tessari M, Heus HA, Wijmenga SS (2012) Fast production of homogeneous recombinant RNA-towards large-scale production of RNA. Nucleic Acids Res 40(13):e102CrossRefGoogle Scholar
  30. Paige JS, Wu KY, Jaffrey SR (2011) RNA mimics of green fluorescent protein. Science 333(6042):642–646CrossRefGoogle Scholar
  31. Paige JS, Nguyen-Duc T, Song W, Jaffrey SR (2012) Fluorescence imaging of cellular metabolites with RNA. Science 335(6073):1194CrossRefGoogle Scholar
  32. Pereira P, Pedro AQ, Tomas J, Maia CJ, Queiroz JA, Figueiras A, Sousa F (2016a) Advances in time course extracellular production of human pre-miR-29b from Rhodovulum sulfidophilum. Appl Microbiol Biotechnol 100(8):3723–3734CrossRefGoogle Scholar
  33. Pereira PA, Tomas JF, Queiroz JA, Figueiras AR, Sousa F (2016b) Recombinant pre-miR-29b for Alzheimer’s disease therapeutics. Sci Rep 6:19946CrossRefGoogle Scholar
  34. Pereira P, Pedro AQ, Queiroz JA, Figueiras AR, Sousa F (2017) New insights for therapeutic recombinant human miRNAs heterologous production: Rhodovolum sulfidophilum vs Escherichia coli. Bioengineered 8(5):670–677CrossRefGoogle Scholar
  35. 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
  36. Ponchon L, Dardel F (2007) Recombinant RNA technology: the tRNA scaffold. Nat Methods 4(7):571–576CrossRefGoogle Scholar
  37. Ponchon L, Beauvais G, Nonin-Lecomte S, Dardel F (2009) A generic protocol for the expression and purification of recombinant RNA in Escherichia coli using a tRNA scaffold. Nat Protoc 4(6):947–959CrossRefGoogle Scholar
  38. Ranaei-Siadat E, Merigoux C, Seijo B, Ponchon L, Saliou JM, Bernauer J, Sanglier-Cianferani S, Dardel F, Vachette P, Nonin-Lecomte S (2014) In vivo tmRNA protection by SmpB and pre-ribosome binding conformation in solution. RNA 20(10):1607–1620CrossRefGoogle Scholar
  39. Rosano GL, Ceccarelli EA (2014) Recombinant protein expression in Escherichia coli: advances and challenges. Front Microbiol 5:172Google Scholar
  40. Schindler D, Dai J, Cai Y (2018) Synthetic genomics: a new venture to dissect genome fundamentals and engineer new functions. Curr Opin Chem Biol 46:56–62CrossRefGoogle Scholar
  41. Sun F, Fu H, Liu Q, Tie Y, Zhu J, Xing R, Sun Z, Zheng X (2008) Downregulation of CCND1 and CDK6 by miR-34a induces cell cycle arrest. FEBS Lett 582(10):1564–1568CrossRefGoogle Scholar
  42. Tu MJ, Ho PY, Zhang QY, Jian C, Qiu JX, Kim EJ, Bold RJ, Gonzalez FJ, Bi H, Yu AM (2019) Bioengineered miRNA-1291 prodrug therapy in pancreatic cancer cells and patient-derived xenograft mouse models. Cancer Lett 442:82–90CrossRefGoogle Scholar
  43. 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
  44. Yamadori T, Ishii Y, Homma S, Morishima Y, Kurishima K, Itoh K, Yamamoto M, Minami Y, Noguchi M, Hizawa N (2012) Molecular mechanisms for the regulation of Nrf2-mediated cell proliferation in non-small-cell lung cancers. Oncogene 31(45):4768–4777CrossRefGoogle Scholar
  45. Yu AM, Jian C, Yu AH, Tu MJ (2019) RNA therapy: are we using the right molecules? Pharmacol Ther 196:91–104CrossRefGoogle Scholar
  46. Zhang X, Potty AS, Jackson GW, Stepanov V, Tang A, Liu Y, Kourentzi K, Strych U, Fox GE, Willson RC (2009) Engineered 5S ribosomal RNAs displaying aptamers recognizing vascular endothelial growth factor and malachite green. J Mol Recognit 22(2):154–161CrossRefGoogle Scholar
  47. Zhang K, Lu X, Li Y, Jiang X, Liu L, Wang H (2019) New technologies provide more metabolic engineering strategies for bioethanol production in Zymomonas mobilis. Appl Microbiol Biotechnol 103(5):2087–2099CrossRefGoogle Scholar
  48. Zhao Y, Tu MJ, Wang WP, Qiu JX, Yu AX, Yu AM (2016) Genetically engineered pre-microRNA-34a prodrug suppresses orthotopic osteosarcoma xenograft tumor growth via the induction of apoptosis and cell cycle arrest. Sci Rep 6:26611CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Biochemistry & Molecular MedicineUC Davis School of MedicineSacramentoUSA

Personalised recommendations