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


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.


Noncoding RNA microRNA siRNA Bioengineering Lung cancer 


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.


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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

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