Nucleoside Optimization for RNAi: A High-Throughput Platform

Abstract

The RNA induced silencing complex (RISC) contains at its core the endonuclease Argonaute (Ago) that allows for guide strand (GS)-mediated sequence-specific cleavage of the target mRNA. Functionalization of the sugar/phosphodiester backbone of the GS, which is in direct contact with Ago, presents a logical opportunity to affect RISC's activity. A systematic evaluation of modified nucleosides requires the synthesis of phosphoramidites corresponding to all four canonical bases (A, U, C, and G) and their sequential evaluation at each position along the 21-nucleotide-long GS. With the use of a platform approach, the sequential replacement of canonical bases with inosine greatly simplifies the problem and defines a new activity baseline toward which the corresponding sugar-modified inosines are compared. This approach was validated using 2'-O-benzyl modification, which demonstrated that positions 5, 8, 15, and 19 can accommodate this large group. Application of this high-throughput methodology now allows for hypothesis-driven rational design of highly potent, immunologically silent and stable siRNAs suitable for therapeutic applications.

siRNAmod: A database of experimentally validated chemically modified siRNAs

“…Both strands were then combined to generate permutations and combinations of cm-siRNAs. Butora et al employed walkthrough method 13 32 , which replaces single or multiple nucleotides in siRNA with different modifications sequentially from one end to another and checking their biological impact. Further effect on enhanced efficacy due to chemical modifications was tested using different siRNAs concentrations 49 .…”

“…For instance phosphorothioate enhances the stability, uptake and biodistribition of the siRNA 30 31 . Large-scale screens and high throughput platforms using different permutation and combinations of chemical modifications were explored to check their effect 24 32 . Native (un-modified) siRNA show immunological activation in vitro to various degrees, while certain chemical modifications (2′-O-methyl) of siRNA avoid immunological effects 33 .…”

siRNA-optimized Modifications for Enhanced In Vivo Activity

“…In previous work, we have also measured the Tm's of the 2'-O-benzyl modifications in the siRNA walkthrough and found no differences at any position between the modified and unmodified siRNAs. 29 …”

“…The 2'-O-benzyl and 2'-O-CH2Py(4) modified phosphoramidites were synthesized using procedures analogous to those described. 29 All nonhydrolytic reactions, unless indicated otherwise were carried out in dry solvents purchased from Aldrich (St. Louis, MO). High-performance liquid chromatography analyses, except for the amidites, were performed at 60°C using an Agilent Zorbax Eclipse Plus C18, 2.1 × 50mm, 1.8 micron column, at 0.8ml/minute flow rate, eluted with a gradient (5–95%) of acetonitrile and water with formic acid (0.1%) as a modifier.…”

“…29 Due to this surprising finding, we decided to further evaluate this new modification for its potential to be used in RNAi therapeutics. In this study we evaluated, in vivo , 2'-O-benzyl, and its pyridine derivative 2'-O-methyl-4-pyridine (2'-O-CH2Py(4)).…”