In RNA interference, small interfering RNAs recognize messenger RNA homologs in cells and induce their degradation.
When developing siRNA-based drugs for therapeutic use, problems encountered are their low efficiency of delivery to targeted cells and degradation by cellular nucleases. A promising approach to improve cellular delivery of siRNA is bioconjugation. Like other oligonucleotides, siRNA conjugation to lipophilic molecules, antibodies, aptamers, ligands, peptides, or polymers enhances cellular delivery and selective targeting.
![](/images/userfiles/image/siRNA mechanism 1.jpg)
Figure 1: Mechanism of systemic and transitive RNAi. Dicer cleavage of dsRNA into 21-23 nt siRNA initiates RNAi. RISC unwinds siRNA duplexes. siRNA binds to mRNA. Either the targeted mRNA is destructed or amplified via RdRP. Amplified dsRNA can transitively silence a 5’-located mRNA in a second cell {Engelke, David R.: RNA interference (RNAi). Nuts and bolts of RNAi technology. DNA Press 2003}.
Table 1: Phosphate Backbone Modifications and their Effect.
Modification
|
Structure
|
ΔTm duplex per modification
|
Impact on the efficiency of RNAi
|
Others
|
Phosphorothioate (PS)
|
![](/images/userfiles/image/PS.jpg)
|
−0.7°C
|
PS inhibits RNAi when introduced in the central part of the antisense strand.
|
PS protects siRNAs from the action of exoribonucleases in vitro and in vivo.
>50% PS cause toxicity in vitro and in vivo.
|
Dimethylethylenediamine (DMEDA)
|
![](/images/userfiles/image/DMEDA.jpg)
|
−0.7–3.4°C
(shown only for thymidine)
|
10% DMEDA in the sense strand are tolerated by RNAi.
|
The effect on nuclease resistance of siRNA is not known.
|
Tert-butyl-S-acyl-2-thioethyl (tBu-SATE)
|
![](/images/userfiles/image/tBuSATE.jpg)
|
No data.
|
25% tBu-SATE are tolerated by RNAi.
|
≥20–40% tBu-SATE in siRNA increase nuclease resistance in vitro and in vivo.
Increases hydrophobicity of siRNA.
Cleaved by thioesterase in the cytoplasm of the cell giving a phosphodiester bond.
|
Boranophosphate (BP)
|
![](/images/userfiles/image/Boronophsophate.jpg)
|
+0.4–1°C (<50% of siRNA)
−0.8–2.5°C (>50% of siRNA)
|
>50% PB inhibit RNAi, the central part of the antisense strand is the most sensitive to modifications.
|
Approximately two times more effectively protect against ribonucleases than PS, but do not cause toxicity in vitro.
|
Amide linker
|
![](/images/userfiles/image/Amide linker.jpg)
|
−0.3 to +0.9°C
|
In some siRNA positions, a single substitution for an amide linker is tolerated by RNAi.
|
The introduction of two amide linkers from the 3′ ends of the duplex increases the nuclease resistance of siRNA in serum.
|
|
|
|
|
|
|
Adapted from: Chernikov IV, Vlassov VV, Chernolovskaya EL. Current Development of siRNA Bioconjugates: From Research to the Clinic. Front Pharmacol. 2019 Apr 26;10:444. doi: 10.3389/fphar.2019.00444. PMID: 31105570; PMCID: PMC6498891. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6498891/.
---...---