Development and Applications of Bioengineered Small Interfering RNA Molecules


Student thesis: Doctoral Thesis

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Awarding Institution
  • Linfeng HUANG (Supervisor)
  • Ying LI (Supervisor)
  • Yung Fu CHANG (External person) (External Co-Supervisor)
Award date7 Jan 2020


RNA interference (RNAi) is a conserved gene silencing mechanism in most eukaryotic cells. When small interfering RNA (siRNA) binds to a messenger RNA (mRNA) with a complementary sequence, the mRNA is degraded by intracellular RNAi machinery. Consequently, the expression of the corresponding gene is transiently silenced. For decades, since RNAi was clearly established, small interfering RNA (siRNA) has been widely employed as a gene silencing tool, and the market demand for siRNA products continues to expand. Among all siRNA products, a novel kind named prokaryotic siRNA (pro-siRNA) stood out in the year of 2013. Produced from recombinant Escherichia coli (E. coli), pro-siRNA obtains plentiful intrinsic merits, such as cost-effective production, impressive gene silencing performance, and negligible off-target effects.

During the period of my PhD studies, I have mainly been devoted to the development and applications of pro-siRNA technology. To prove the potency of pro-siRNA technology, several pro-siRNAs were designed to target key genes of the Ebola virus in an analog virus model. Preliminary results showed the significant knockdown efficiency of the home-made pro-siRNAs. To expand the dimension of pro-siRNA technology, a system for high-throughput production and screening was established. To confirm a wide application range of the pro-siRNA library produced by the system, two loss-of-function transcriptome-wide screens were performed to target genes regulating exogenous gene expression and those essential for cancer cell survival, respectively.

In the first screen of 576 pro-siRNAs derived from HeLa-EGFP cell line, besides the successful targeting of the EGFP gene as the most direct gene to affect EGFP expression, genes from SUMOylation pathway have also been identified as a group of indirect, whereas key regulator genes for exogenous EGFP expression. A library of 1056 FDA approved compounds were applied to the same cell line as a supplementary exploration for any correlation between SUMOylation mediated EGFP expression and drug effects, and SUTENT was identified to significantly increase GFP signal after introduction to the GFP-expressing cell line. Validation experiments showed an alteration of the global SUMOylation pattern after SUTENT treatment.

The second screen of 1920 pro-siRNAs targeting the cervical cancer cell line HeLa recognized 72 gene candidates as essential for cancer cell viability. 71 out of the 72 candidates were also identified by previous genome-wide RNAi or CRISPR screens, suggesting the low false positive rate of screens using the pro-siRNA library.

The results of both screens largely correlated with previous works by other screening projects, thus demonstrating the consistent, reliable quality of the pro-siRNA libraries. The successful establishment of pro-siRNA high-throughput system contributes to the diversity of RNAi technologies and offers more screening strategies to RNAi application users.