Amide linkages mimic phosphates in RNA interactions with proteins and are well tolerated in the guide strand of short interfering RNAs

Abstract

While the use of RNA interference (RNAi) in molecular biology and functional genomics is a well-established technology, in vivo applications of synthetic short interfering RNAs (siRNAs) require chemical modifications. We recently found that amides as non-ionic replacements for phosphodiesters may be useful modifications for optimization of siRNAs. Herein, we report a comprehensive study of systematic replacement of a single phosphate with an amide linkage throughout the guide strand of siRNAs. The results show that amides are surprisingly well tolerated in the seed and central regions of the guide strand and increase the silencing activity when placed between nucleosides 10 and 12, at the catalytic site of Argonaute. A potential explanation is provided by the first crystal structure of an amide-modified RNA–DNA with Bacillus halodurans RNase H1. The structure reveals how small changes in both RNA and protein conformation allow the amide to establish hydrogen bonding interactions with the protein. Molecular dynamics simulations suggest that these alternative binding modes may compensate for interactions lost due to the absence of a phosphodiester moiety. Our results suggest that an amide can mimic important hydrogen bonding interactions with proteins required for RNAi activity and may be a promising modification for optimization of biological properties of siRNAs.

Synthesis and Biological Activity of Short Interfering RNAs Having Several Consecutive Amide Internucleoside Linkages

“…Increasing siRNA duplex concentrations to 100 n m somewhat improved the silencing activity to 35 %, 25 %, and 90 % for G3 , G4 , and G0 , respectively (Figure S14). This result was consistent with our earlier study that showed that isolated amide linkages at most internal positions of this guide strand strongly reduce its RNAi activity. We later showed that the decreased silencing activity was due to preferential loading of the unmodified passenger P0 (instead of the modified guides) into Ago2, and that the activity could be restored by using passenger‐strand P1 , having a single amide linkage between its first and second nucleosides.…”

“…The G0 – P0 duplex was rich in Us and As at the 3'‐end of the guide and 5'‐end of the passenger strand, which made synthesis of amide‐linked RNAs easier because only modified U and A monomers were needed. Our previous studies showed that both guide and passenger strands of this siRNA were highly active (the passenger was also loaded in Ago2) and that isolated amide modifications in the guide strand strongly reduced its activity when paired with an unmodified passenger strand. Hence, G0 – P0 was a good model system due to its relatively easy synthesis and high sensitivity to amide modification, which would provide a rigorous test of the limits of consecutive amide modifications that may be tolerated in an siRNA.…”

“…We have previously reported that isolated amide linkages did not significantly change the structure and thermal stability of RNA duplexes and caused relatively small decreases in RNAi activity when incorporated in an siRNA guide strand . Three consecutive amide linkages caused relatively small changes in the structure of an A‐type duplex; however, the melting temperature of the duplex was significantly lowered .…”

“…In the present study, we used our recently developed and more efficient synthesis route that gave 2 a in only six steps and 31 % overall yield and 2 b in eight steps and 16 % overall yield . Switch monomers 1 a and 1 b were prepared as previously reported . The synthesis of monomers 3 and 4 (for details, see Supporting Information) started with a non‐selective TOM protection of 5'‐azidouridine (Scheme ) that gave a separable mixture of 3'‐ and 2'‐ O ‐TOM isomers 5 and 6 , respectively.…”

“…The unmodified RNA parts of all sequences were synthesized by standard phosphoramidite chemistry using 2'‐ O ‐TOM protected monomers (Glen Research) on an Expedite 8909 DNA/RNA synthesizer. The amide‐modified guide and passenger strands were cleaved from the solid support, deprotected, purified, and analyzed (MALDI‐TOF MS) using standard RNA synthesis protocols, as recommended by Glen Research and previously reported by us . Overall, the modest amide coupling yields enabled synthesis of sufficient amounts of modified RNA strands having up to six consecutive amide linkages (for yields, see Table S1 and Figures S1–S12).…”