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Argonaute-2 can bind metabolic stable siRNAs

Incorporating chemical modifications will stabilize small interfering RNAs (siRNAs) metabolically and improve tissue distribution.

RNA interference (RNAi) is an endogenous regulatory pathway silencing a gene in a sequence-specific manner. However, a significant barrier for efficient gene silencing using RNAi is the difficulty of delivering small interfering RNAs (siRNAs) in vivo. Design strategies addressing low stability and non-targeted biodistribution are needed. Also, therapeutic siRNAs must avoid the stimulation of undesirable innate immune responses.

Gavrilov & Saltzman in 2012 reviewed the mechanistic principles of RNA interference and its use in biomedical applications. This review also described the use of chemical modifications for siRNA sugars and backbone, including bridged nucleic acids (BNAs) and others. 

In 2016, Schirle et al. solved the crystal structure of human Argonaute-2 (Ago2) bound to a modified, metabolically stable siRNA guide sequence with extensive backbone modifications to show that Argonaute-2 can bind pharmacologically stable siRNAs.

The comparison of a complex with an un-modified siRNA and modified siRNA guide revealed that the Ago2 structure is relatively unaffected by chemical modifications in the bound siRNA. According to Schirle et al., the modified siRNA appeared to be more plastic and shifts, relative to the unmodified siRNA, to optimize contacts with Ago2. The structural study revealed that even significant conformational perturbations in the 3′-half of the siRNA seed region have a relatively modest effect on knock-down potency. These findings explained why various modification patterns are tolerated in siRNAs and revealed the structural basis for new therapeutic siRNA designs.

RNA modifications used by Schile et al. are shown in figure 1.



Figure 1: Structures with RNA modifications. Blue: 2′-O-Me, Green: 2′-F, purple: 2′-O-MOE, red: VP-T = 2′-O-MOE-thymidine-(E)-5′- vinylphosphonate; s: phosphorothioate. All backbone linkages are phosphodiesters except those indicated with s (Adapted from Schirle et al.).

Example of a modified siRNA sequence used in the study:

VP-TsUsAUsCUsAUsAAsUGsAUsCsAsGsGsUsAsA

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Figure 2; Various views of a Human Argonaute2-siRNA complex.

Reference

Gavrilov K, Saltzman WM. Therapeutic siRNA: principles, challenges, and strategies. Yale J Biol Med. 2012 Jun;85(2):187-200. Epub 2012 Jun 25. PMID: 22737048; PMCID: PMC3375670. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3375670/

Schirle NT, Kinberger GA, Murray HF, Lima WF, Prakash TP, MacRae IJ. Structural Analysis of Human Argonaute-2 Bound to a Modified siRNA Guide. J Am Chem Soc. 2016 Jul 20;138(28):8694-7. doi: 10.1021/jacs.6b04454. Epub 2016 Jul 12. PMID: 27380263; PMCID: PMC4993527.

https://www.rcsb.org/structure/5JS2

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