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Draft:ShRNA Design Strategies

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  • Comment: My concern is that it relies only on primary sources, research into shRNAs, and may be too technical/advanced for a general encyclopaedia. I am not opposed to this article being created, but same as Ozzie, I'm not sure. TurboSuperA+(talk) 05:24, 23 July 2025 (UTC)
  • Comment: seems interesting/notable, however other editors should take a look Ozzie10aaaa (talk) 18:20, 21 July 2025 (UTC)

Short hairpin RNAs (shRNAs) are synthetic RNA molecules widely used for gene silencing via the RNA interference (RNAi) pathway.[1][2] Over the years, different generations and strategies of shRNA design have emerged to improve their processing, reduce off-target effects, and increase efficiency.[3][4]

First and second generation shRNAs

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First-generation shRNAs, as described by Silva et al. (2005) and Cheng et al. (2006), are typically expressed under RNA polymerase III (Pol III) promoters such as U6 or H1.[5] These shRNAs are processed by Dicer into siRNA-like duplexes. Second-generation shRNAs incorporate a miRNA scaffold, such as the miR-30 backbone, and can be expressed under both Pol III and RNA polymerase II (Pol II) promoters (e.g., CMV).[6] The addition of miRNA flanking sequences improves processing efficiency and mimics natural miRNA biogenesis.[7]

Asymmetry design

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Ding et al. (2007) introduced asymmetrical designs to improve strand selection during RISC loading.[8][9] This strategy promotes incorporation of the intended guide strand and reduces off-target effects caused by the passenger strand.[9]

Loop Position optimization

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Gu et al. (2012) demonstrated that the position and composition of the loop region in shRNAs or pre-miRNAs significantly affects Dicer processing and overall silencing efficiency.[10]

Organic shRNAs (OshRNAs)

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Zeng et al. (2013) developed organic small hairpin RNAs (OshRNAs) that mimic endogenous miRNAs,[11] these constructs incorporate bulges and mismatches to:

  • Enhance guide strand accumulation
  • Suppress passenger strand loading
  • Target the 3′ untranslated region (3′UTR) of mRNA

These features help improve silencing efficacy while minimizing off-target effects.[11]

Optimal design factors

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Bofill-De Ros and Gu (2016) outlined essential parameters for optimizing shRNA design under both Pol II and Pol III promoters,[12] for Pol III systems:

  • Initiation with a Guanosine (G) improves transcription efficiency.
  • A poly-T (4–5 T residues) serves as a termination signal.
  • The 5′ end of the guide strand should be less thermodynamically stable than the 5′ end of the passenger strand to favor correct RISC incorporation.[12]

Third generation: AgoshRNAs

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AgoshRNAs are Dicer-independent, third-generation shRNAs processed by Ago2, bypassing Dicer cleavage. These are typically expressed under Pol II promoters and offer precise guide strand generation with reduced off-target effects.[13]

Artificial Third generation designs: miR-E and miR-3G

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Watanabe et al. (2016) introduced artificial variants like miR-E and miR-3G, combining high expression with efficient processing in mammalian cells.[14]

Periodic shRNAs (p-shRNAs) and open-ended p-shRNAs

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As of 2024, periodic shRNAs (p-shRNAs) have been synthesized via rolling circle transcription of circular DNA templates. Open-ended p-shRNAs (op-shRNAs), generated by selective enzyme digestion, significantly enhance functional siRNA processing—showing over tenfold higher efficiency compared to traditional shRNAs.[15]

References

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  1. ^ Dana, H; Chalbatani, GM; Mahmoodzadeh, H; Karimloo, R; Rezaiean, O; Moradzadeh, A; Mehmandoost, N; Moazzen, F; Mazraeh, A; Marmari, V; Ebrahimi, M; Rashno, MM; Abadi, SJ; Gharagouzlo, E (June 2017). "Molecular Mechanisms and Biological Functions of siRNA". International Journal of Biomedical Science : IJBS. 13 (2): 48–57. doi:10.59566/IJBS.2017.13048. PMC 5542916. PMID 28824341.
  2. ^ Fire, Andrew; Xu, SiQun; Montgomery, Mary K.; Kostas, Steven A.; Driver, Samuel E.; Mello, Craig C. (February 1998). "Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans". Nature. 391 (6669): 806–811. Bibcode:1998Natur.391..806F. doi:10.1038/35888. PMID 9486653.
  3. ^ Moore, CB; Guthrie, EH; Huang, MT; Taxman, DJ (2010). "Short Hairpin RNA (ShRNA): Design, Delivery, and Assessment of Gene Knockdown". RNA Therapeutics. Methods in Molecular Biology (Clifton, N.J.). Vol. 629. pp. 141–58. doi:10.1007/978-1-60761-657-3_10. ISBN 978-1-60761-656-6. PMC 3679364. PMID 20387148.
  4. ^ McIntyre, Glen J; Fanning, Gregory C (December 2006). "Design and cloning strategies for constructing shRNA expression vectors". BMC Biotechnology. 6 (1) 1. doi:10.1186/1472-6750-6-1. PMC 1343552. PMID 16396676.
  5. ^ Silva, JM; Li, MZ; Chang, K; Ge, W; Golding, MC; Rickles, RJ; Siolas, D; Hu, G; Paddison, PJ; Schlabach, MR; Sheth, N; Bradshaw, J; Burchard, J; Kulkarni, A; Cavet, G; Sachidanandam, R; McCombie, WR; Cleary, MA; Elledge, SJ; Hannon, GJ (November 2005). "Second-generation shRNA libraries covering the mouse and human genomes". Nature Genetics. 37 (11): 1281–8. doi:10.1038/ng1650. PMID 16200065.
  6. ^ Zhou, H. (23 March 2005). "An RNA polymerase II construct synthesizes short-hairpin RNA with a quantitative indicator and mediates highly efficient RNAi". Nucleic Acids Research. 33 (6): e62. doi:10.1093/nar/gni061. PMC 1074311. PMID 15805121.
  7. ^ Bernards, R; Brummelkamp, TR; Beijersbergen, RL (September 2006). "shRNA libraries and their use in cancer genetics". Nature Methods. 3 (9): 701–6. doi:10.1038/nmeth921. PMID 16929315.
  8. ^ Kutter, Claudia; Svoboda, Petr (October 2008). "miRNA, siRNA, piRNA: Knowns of the unknown". RNA Biology. 5 (4): 181–188. doi:10.4161/rna.7227. PMID 19182524.
  9. ^ a b Ding, H; Liao, G; Wang, H; Zhou, Y (15 August 2007). "Asymmetrically designed siRNAs and shRNAs enhance the strand specificity and efficacy in RNAi". Journal of RNAi and Gene Silencing : An International Journal of RNA and Gene Targeting Research. 4 (1): 269–80. PMC 2737237. PMID 19771234.
  10. ^ Gu, Shuo; Jin, Lan; Zhang, Yue; Huang, Yong; Zhang, Feijie; N. Valdmanis, Paul; A. Kay, Mark (November 2012). "The Loop Position of shRNAs and Pre-miRNAs Is Critical for the Accuracy of Dicer Processing In Vivo". Cell. 151 (4): 900–911. doi:10.1016/j.cell.2012.09.042. PMC 3499986. PMID 23141545.
  11. ^ a b Zeng, Mei; Kuzirian, Marissa S.; Harper, Lamia; Paradis, Suzanne; Nakayama, Takuya; Lau, Nelson C. (September 2013). "Organic small hairpin RNAs (OshR): A do-it-yourself platform for transgene-based gene silencing". Methods. 63 (2): 101–109. doi:10.1016/j.ymeth.2013.05.007. PMC 3966114. PMID 23707624.
  12. ^ a b Bofill-De Ros, X; Gu, S (1 July 2016). "Guidelines for the optimal design of miRNA-based shRNAs". Methods (San Diego, Calif.). 103: 157–66. doi:10.1016/j.ymeth.2016.04.003. PMC 4921303. PMID 27083402.
  13. ^ Herrera-Carrillo, E; Harwig, A; Berkhout, B (August 2017). "Silencing of HIV-1 by AgoshRNA molecules". Gene Therapy. 24 (8): 453–461. doi:10.1038/gt.2017.44. PMID 28553929.
  14. ^ Watanabe, C; Cuellar, TL; Haley, B (2016). "Quantitative evaluation of first, second, and third generation hairpin systems reveals the limit of mammalian vector-based RNAi". RNA Biology. 13 (1): 25–33. doi:10.1080/15476286.2015.1128062. PMC 4829305. PMID 26786363.
  15. ^ Saw, Phei Er; Song, Erwei (2025). "RNA Nanotechnology: Biomedical Application". RNA Therapeutics in Human Diseases. pp. 565–589. doi:10.1007/978-981-96-3041-7_24. ISBN 978-981-96-3040-0.
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