Amide Modifications in the Seed Region of the Guide Strand Improve the On-Target Specificity of Short Interfering RNA

Abstract

RNA interference (RNAi) is a well-established research tool and is also maturing as a novel therapeutic approach. For the latter, microRNA-like off-target activity of short interfering RNAs (siRNAs) remains as one of the main problems limiting RNAi drug development. In this communication, we report that replacement of a single internucleoside phosphodiester in the seed region (nucleotides 2 to 7) of the guide strand with an amide linkage suppressed the undesired microRNA-like off-target activity by at least an order of magnitude. For the specific siRNA targeting the PIK3CB gene, an amide modification between the third and fourth nucleotides of the guide strand showed the strongest enhancement of specificity (completely eliminated off-target silencing) while maintaining high on-target activity. These results are important because off-target activity is one of the main remaining roadblocks for RNA based drug development.

Synthesis and Properties of RNA Modified with Cationic Amine Internucleoside Linkage

“…target activity, which was similar to the effect of amide linkages at these positions in our previous study. 19 At these positions, amine modifications caused a small decrease in activity and specificity compared to the nonmodified and amide-modified siRNAs.…”

“…Our research group has focused on amides and other non-phosphorus linkages as replacements for the phosphodiester backbone in RNA. , Our early studies showed that amides, especially AM1 (Figure ), were excellent structural mimics of phosphates in RNA duplexes. , Related studies showed that the formacetal linkage (FA) was an almost perfect structural mimic of phosphate in RNA and suggested that nonionic backbone modifications were in general better accommodated in RNA than in DNA sequences. , Follow-up studies showed that amides as backbone modifications were well-tolerated in siRNAs , and when placed at specific positions significantly reduced the off-target activity of passenger or guide strands of siRNAs . Amides were also tolerated in CRISPR-associated RNAs and did not reduce Cas9 activity when placed in the PAM-distal region of the guide strand .…”

“…To evaluate the effect of amine linkages on siRNA activity and specificity, we used a dual luciferase assay of siRNA targeting human PIK3CB mRNA (positions 998 to 1017). This well-established assay , was also used in our previous study on amide-modified siRNAs . The assay compares on-target activity for PIK3CB with silencing of two off-target mRNAs, YY1 and FADD, to evaluate the effect of chemical modifications on siRNA activity and specificity (Figure S11 and Table S5).…”

Synthesis of Dinucleotide Non‐Symmetrical Triester Phosphate Phosphoramidites and Their Incorporation Within Oligonucleotides

“…However, these groups still retain the negative charge. Neutral groups such as amides, triazoles, and others have been successfully used as neutral backbone mimics (Efthymiou et al., 2012; Richter et al., 2023). Increasing the repertoire of neutral backbone modifications is necessary to overcome the aforementioned problems.…”

Synthesis, Duplex-Forming Ability, and Enzymatic Stability of Oligonucleotides Modified with Amide-Linked Dinucleotides Containing a 3′,4′-Tetrahydropyran-Bridged Nucleic Acid

“…Replacement of a phosphodiester linkage in oligonucleotides by an amide linkage can increase their binding affinity with RNA . Moreover, oligonucleotides, which have partial amide linkages, are resistant to degradation by nuclease compared to unmodified DNA; therefore, antisense oligonucleotides and siRNAs with amide linkages have already been applied for some in vitro and in vivo studies. − …”