Part:BBa_K5062017
Anti-PD1 Short Hairpin RNA (shRNA) Release System
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 81
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Overview
This is a release system designed for the release of anti-PD1 short hairpin RNA (shRNA) (BBa_K5062012) to allow for RNA inteference operations (RNAi) to disrupt the expression of PD1. The system utilizes a 5' Hammerhead Ribozyme (BBa_K5062009) and a 3' Hepatitis Delta Virus (HDV) (BBa_K5062010) Ribozyme to catalytically self-cleave any unnecessary sequences at 5' and 3' ends of the shRNA allowing for its exact sequence to be preserved.
This system allows shRNA expression using any RNA polymerase, including those that inherently contain capping and tailing capabilities such as the RNA polymerase of Venezuelan equine encephalitis (VEE) virus which powers self-replicating RNA (srRNA) systems, allowing a transient srRNA system to sustain the subgenomic expression and release of base-perfect shRNA.
In vitro characterisation of this part is currently ongoing prior to the wiki freeze. Full part characterisation is expected by the 2024 iGEM Giant Jambouree.
Usage and Biology
Short Hairpin RNA (shRNA) is indeed a single-stranded RNA that folds back on itself forming a stem-loop structure. This loop is what gives the shRNA the appearance of a double-stranded RNA (dsRNA). The non-complementary region of the RNA sequence forms the loop that connects the two ends of the stem, allowing it to fold back on itself.
Once the shRNA is introduced into the host cell, the shRNA is transcribed by its promoter. The transcribed shRNA forms a stem-loop structure which is then transported from the nucleus to the cytoplasm by the action of Exportin 5. Then it is processed by an endoribonuclease known as “DICER” which cleaves the loop and converts the shRNA into short dsRNAs or small interfering RNA (siRNA).
Upon getting processed by DICER, the dsRNA is then loaded into the RNA-induced silencing complex (RISC), where the sense (passenger) strand gets degraded, leaving the antisense (guide) strand to guide the RISC complex towards the complementary target mRNA sequence (O’Keefe, 2013). A crucial component of RISC that plays a central role in the gene silencing mechanism is the presence of Argonaute 2 (Ago2) protein in RISC. Ago2 possesses endonuclease activity which allows it to cleave the target mRNA at the target site when the guide strand perfectly complements the target strand (Ruda et al., 2014). This cleavage results in the degradation of the target mRNA, leading to effective silencing of the target gene.
In our case, the guide strand of the designed shRNA binds to the mRNA of SIRPα leading to its cleavage and degradation by RISC. This cleavage of SIRPα results in a significant reduction in the expression levels of SIRPα which enhances the phagocytic activity. As the blockage of the PD1-PDL1 pathway leads to a significant increase in phagocytotic ability (Feng et al., 2019).
A simplified diagram depicting the processing of shRNA in vivo leading to transcriptional regulation (Fouquerel et al., 2014)
The self-cleavage reaction catalyzed by the Hammerhead Ribozyme involves a phosphodiester isomerization mechanism. This results in the formation of a 2',3'-cyclic phosphate at the 5' end and a 5'-hydroxyl group at the 3' end of the cleaved RNA. The reaction is reversible, allowing the Hammerhead Ribozyme to also catalyze the ligation of the cleaved RNA strands (Hammann et al., 2012).
The Hammerhead Ribozyme is considered one of the best-characterized ribozymes. Its small size, well-studied cleavage chemistry, known crystal structure, and biological relevance make it a useful model system for understanding the fundamental mechanisms of RNA catalysis. Hammerhead Ribozymes have been found ubiquitously across all domains of life, where they play roles in processes like rolling-circle replication of viral genomes and co-transcriptional processing of retrotransposons (Jimenez et al., 2015).
The sequence is only the core of the hammerhead ribozyme. A 6 nucleotide head should be added to the 5' end of the ribozyme core which is complementary to the first 6 nucleotides of the RNA to be released to allow a succesful cleavage. Since this is a DNA part, it should be added to the DNA that eventually transcirbes into the relevant RNA sequences.
A diagram depics the design considerations required for using the 5' Hammerhead Ribozyme (UC San Diego, n.d.)
The HDV ribozyme has a characteristic nested double-pseudoknot secondary structure with five helical regions (P1, P1.1, P2, P3, P4) (Ferré-D’Amaré & Scott, 2010). The HDV ribozyme catalyzes a site-specific transesterification reaction, where the 2' hydroxyl group of the nucleotide upstream of the cleavage site acts as a nucleophile to attack the adjacent phosphodiester bond. This results in the formation of a 2'-3' cyclic phosphate and a 5' hydroxyl product (Jimenez et al., 2015).
The HDV ribozyme can function through both metal-dependent and metal-independent mechanisms. In the metal-dependent mechanism, a divalent metal ion (typically Mg2+) helps to stabilize the developing negative charge on the 2' nucleophile and the pentavalent phosphorane transition state (Ke et al., 2004). In the metal-independent mechanism, the catalytic cytosine (C75) can act as a general acid to protonate the 5' leaving group (Ferré-D’Amaré & Scott, 2010).
The self-cleavage activity of the HDV ribozyme is essential for processing the HDV genomic and antigenomic RNA strands during rolling-circle replication (Ferré-D'Amaré, A.R et. al,2010). The cleavage generates linear RNA strands with 5' hydroxyl and 2'-3' cyclic phosphate ends, which can then be ligated to form the circular HDV genome (Jimenez, R.M. et al., 2015).
This can be added directly to the 3' end of RNA sequences. Since this is a DNA part, it should be added to the DNA that eventually transcirbes into the relevant RNA sequences.
A diagram depics the design considerations required for using the 3' HDV Ribozyme (UC San Diego, n.d.)
References
Feng, M., Jiang, W., Kim, B. Y. S., Zhang, C. C., Fu, Y., & Weissman, I. L. (2019). Phagocytosis checkpoints as new targets for cancer immunotherapy. Nature Reviews. Cancer, 19(10), 568–586. https://doi.org/10.1038/s41568-019-0183-z
Ferré-D’Amaré, A. R., & Scott, W. G. (2010). Small self-cleaving ribozymes. Cold Spring Harbor Perspectives in Biology, 2(10), a003574. https://doi.org/10.1101/cshperspect.a003574
Fouquerel, E., Li, J., Braganza, A., Yu, Z., Brown, A. R., Wang, X., Schamus, S., Svilar, D., Fang, Q., & Sobol, R. W. (2014). Use of RNA interference to study DNA repair. In Methods in pharmacology and toxicology (pp. 413–447). https://doi.org/10.1007/978-1-4939-1068-7_24
Hammann, C., Luptak, A., Perreault, J., & De La Peña, M. (2012). The ubiquitous hammerhead ribozyme. RNA, 18(5), 871–885. https://doi.org/10.1261/rna.031401.111
Jimenez, R. M., Polanco, J. A., & Lupták, A. (2015). Chemistry and Biology of Self-Cleaving Ribozymes. Trends in Biochemical Sciences, 40(11), 648–661. https://doi.org/10.1016/j.tibs.2015.09.001
Ke, A., Zhou, K., Ding, F., Cate, J. H. D., & Doudna, J. A. (2004). A conformational switch controls hepatitis delta virus ribozyme catalysis. Nature, 429(6988), 201–205. https://doi.org/10.1038/nature02522
O’Keefe, E. P. (2022, October 29). SiRNAs and SHRNAs: Tools for protein knockdown by gene Silencing. https://www.labome.com/method/siRNAs-and-shRNAs-Tools-for-Protein-Knockdown-by-Gene-Silencing.html
Ruda, V. M., Chandwani, R., Sehgal, A., Bogorad, R. L., Akinc, A., Charisse, K., Tarakhovsky, A., Novobrantseva, T. I., & Koteliansky, V. (2014). The roles of individual mammalian argonautes in RNA interference in vivo. PLoS ONE, 9(7), e101749. https://doi.org/10.1371/journal.pone.0101749
UC San Diego. (n.d.). 3’ Ribozyme design. https://labs.biology.ucsd.edu/zhao/CRISPR_web/3_prime_ribozyme_design.html
UC San Diego. (n.d.). 5’ Ribozyme design. https://labs.biology.ucsd.edu/zhao/CRISPR_web/5_prime_ribozyme_design.html
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