Amides are excellent mimics of phosphate internucleoside linkages and are well tolerated in short interfering RNAs

Abstract

RNA interference (RNAi) has become an important tool in functional genomics and has an intriguing therapeutic potential. However, the current design of short interfering RNAs (siRNAs) is not optimal for in vivo applications. Non-ionic phosphate backbone modifications may have the potential to improve the properties of siRNAs, but are little explored in RNAi technologies. Using X-ray crystallography and RNAi activity assays, the present study demonstrates that 3′-CH2-CO-NH-5′ amides are excellent replacements for phosphodiester internucleoside linkages in RNA. The crystal structure shows that amide-modified RNA forms a typical A-form duplex. The amide carbonyl group points into the major groove and assumes an orientation that is similar to the P–OP2 bond in the phosphate linkage. Amide linkages are well hydrated by tandem waters linking the carbonyl group and adjacent phosphate oxygens. Amides are tolerated at internal positions of both the guide and passenger strand of siRNAs and may increase the silencing activity when placed near the 5′-end of the passenger strand. As a result, an siRNA containing eight amide linkages is more active than the unmodified control. The results suggest that RNAi may tolerate even more extensive amide modification, which may be useful for optimization of siRNAs for in vivo applications.

Therapeutic potential of chemically modified siRNA: Recent trends

“…Chemical modifications of phosphodiester linkages with amide modification ( 16 ) using U AM1 U and U AM1 A had been investigated by our group (selvam et al . & Mutisya et al …”

“…The internucleotide phosphodiester linkages of unmodified siRNAs were negatively charged at physiological pH and can be cleaved by endo-and exonucleases. Chemical modifications of phosphodiester linkages with amide modification (16) using U AM1 U and U AM1 A had been investigated by our group (selvam et al [32] & Mutisya et al [33] ). 1-5 amide modifications were introduced in the sense and the antisense strand.…”

Chimeric RNA Oligonucleotides with Triazole and Phosphate Linkages: Synthesis and RNA Interference

“…As is typical with artificial oligonucleotides,– the modification in the passenger strand was well tolerated to achieve siRNA activity, and the passenger strand 9‐i with a triazole modification at 3–4 counting from the 5′‐end interfered the target gene at 7 % expression level. This interference level was comparable with that of the siRNA‐b with natural oligonucleotide 9‐ nat of 4 % expression level.…”

“…As was the case with RNA‐a, the triazole linkers were favorably tolerated in the downstream region, and modifications in the 3′‐overhang regions at 19–20 and 20–21 counting from the 5′‐end with 9‐iii and 9‐iv suppressed the expression levels to 10 % and 6 %, respectively. Because the interference with the 7‐i / 8‐vii combination (Figure S53) was not more potent than those of 9‐i / 10‐ nat or 9‐ nat / 10‐iv combinations, the chimera/chimera combination of two modified oligonucleotides with a triazole linker may not be effective for a synergetic improvement of interference –. However, the modifications in the upstream region considerably affected the siRNA activity.…”

Knockdown of STAT3 expression in SKOV3 cells by biodegradable siRNA–PLGA/CSO conjugate micelles

“…In recent years, many disciplines, including genomics, cell and molecular biology, and material science, have worked on methods for chemical conjugation of siRNA [14,15], such as using cleavable disulfide bonds [16,17], amide linkages [18], or acid labile linkages [19]. In particular, utilizing disulfide bond formation of the complex has been very attractive to researchers.…”