Development of modified siRNA molecules incorporating 5-fluoro-2'-deoxyuridine residues to enhance cytotoxicity

Abstract

Therapeutic small interfering RNAs (siRNAs) are composed of chemically modified nucleotides, which enhance RNA stability and increase affinity in Watson–Crick base pairing. However, the precise fate of such modified nucleotides once the siRNA is degraded within the cell is unknown. Previously, we demonstrated that deoxythymidine release from degraded siRNAs reversed the cytotoxicity of thymidylate synthase (TS)-targeted siRNAs and other TS inhibitor compounds. We hypothesized that siRNAs could be designed with specific nucleoside analogues that, once released, would enhance siRNA cytotoxicity. TS-targeted siRNAs were designed that contained 5-fluoro-2′-deoxyuridine (FdU) moieties at various locations within the siRNA. After transfection, these siRNAs suppressed TS protein and messenger RNA expression with different efficiencies depending on the location of the FdU modification. FdU was rapidly released from the siRNA as evidenced by formation of the covalent inhibitory ternary complex formed between TS protein and the FdU metabolite, FdUMP. These modified siRNAs exhibited 10–100-fold greater cytotoxicity and induced multiple DNA damage repair and apoptotic pathways when compared with control siRNAs. The strategy of designing siRNA molecules that incorporate cytotoxic nucleosides represents a potentially novel drug development approach for the treatment of cancer and other human diseases.

Modified miR-15a has therapeutic potential for improving treatment of advanced stage colorectal cancer through inhibition of BCL2, BMI1, YAP1 and DCLK1

“…The data presented here supports the viability of a novel modification in which 5-FU is incorporated into a miRNA to enhance its cytotoxicity for therapeutic function. This concept has been investigated in siRNA, with the incorporation of 5-fluoro-2’-deoxyuridine into siRNA against TS [ 50 ]. This study also demonstrated enhanced cytotoxicity of the modified siRNA.…”

Rhizoplane Bacteria and Plant Species Co-determine Phosphorus-Mediated Microbial Legacy Effect

“…OTUs were then clustered at the 97% sequence similarity level using the Usearch program that provides clustering, chimera checking, and quality filtering in QIIME. Finally, the most abundant sequence for each OTU was selected as the representative OTU and taxonomic annotations were assigned to each OTU's representative sequence against the SILVA (SSU117/119) 16S rRNA database for bacteria (Yilmaz et al, 2014).…”

Therapeutic potential of chemically modified siRNA: Recent trends

“…5‐Fluoro‐2′‐deoxyuridine (FdU) (Figure ) ( 21 ) moieties were introduced in multiple positions of siRNA targeting thymidylate synthase (TS). The cytotoxicity level of the modified siRNA was improved up to 10‐ to 100‐fold compared to the unmodified siRNA (Table ) . siRNAs modified with 2′‐ O ‐methyl phosphorodithioate (2′ O‐MePS2) ( 22 ) were reported to have effective therapeutic targeting.…”

“…The cytotoxicity level of the modified siRNA was improved up to 10-to 100-fold compared to the unmodified siRNA ( Table 3). [39] siRNAs modified with 2′-O-methyl phosphorodithioate (2′ O-MePS2) (22) were reported to have effective therapeutic targeting. This was confirmed by targeting this modified siRNA against GRAMD 1B gene.…”