Synthesis and in vitro assessment of chemically modified siRNAs targeting BCL2 that contain 2′-ribose and triazole-linked backbone modifications

Abstract

Short-interfering RNAs (siRNAs) are naturally occurring biomolecules used for post-transcriptional gene regulation, and therefore hold promise as a future therapeutic by silencing gene expression of overexpressed deleterious genes. However, there are many inherent problems with the native RNA structure. This project investigates the ability of a library of chemically modified siRNAs to target the therapeutically relevant oncogene, BCL2, by combining 2′-ribose sugar modifications with a novel triazole-linked backbone modification, previously described by our group. Solid support phosphoramidite chemistry was used to incorporate chemical modifications at various positions within anti-BCL2 siRNAs. In vitro effects were evaluated through qPCR, cell viability, nuclease stability, and an ELISA assay. Our results indicate that these unique modifications are well tolerated within RNAi and show enhanced activity and stability over natural siRNAs, while also improving upon inherent issues of toxicity and immunological activity.Med. Chem. Commun. This journal is † Electronic supplementary information (ESI) available: Full list of siRNAs synthesised and assessed as well as detailed experimental materials and methods can be found in the electronic supplementary information. See

Therapeutic Potential of OMe-PS-miR-29b1 for Treating Liver Fibrosis

“…51 Double-stranded RNAs are known to stimulate innate immunity in vivo by triggering pro-inflammatory cytokine release. 52 The role of IFN-g is not clear in liver fibrosis. Several reports conclude that its elevated concentration represents a characteristic feature of liver disease severity in cirrhosis patients and pre-clinical models.…”

Effective gene-silencing of siRNAs that contain functionalized spacer linkages within the central region

“…21,22 Prior work from our research group identied siRNAs bearing triazole-backbone modications were suitable gene silencing substrates for the endogenous gene BCL2. 23 To our delight, siRNAs containing either of these aromatic groups were well accommodated within the central region of the siRNA, and exhibited gene-silencing potency comparable to wild-type wtBcl2.…”

Effective carrier-free gene-silencing activity of sphingosine-modified siRNAs

“…Furthermore, prior studies have shown that triazole functional groups are well tolerated within siRNAs. 32,33…”

“…Furthermore, prior studies have shown that triazole functional groups are well tolerated within siRNAs. 32,33 The resulting compound 9 was phosphitylated with 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite to yield the phosphoramidite derivative 10, which was used for the solidphase oligonucleotide synthesis as described above. The modi-fication was placed at the 3′-end of the sense and antisense strand, replacing the 3′-dTdT overhang, and within an internal uridine nucleotide position on the sense strand.…”