Abstract
The high Ago2 affinity of siRNAs with combined 2′-O-methyl and phosphorodithioate backbone modifications (MePS2) in the 3′-terminal region of the sense strand is likely the result of enhanced hydrophobic interactions with the protein's PAZ domain.
Highlights
Unlike the phosphorothioate moiety (PS; Fig. 1), PS2 is achiral, precluding potential drawbacks that may arise from chiral Psubstituted oligonucleotides, owing to variable biophysical and biochemical properties of individual (e.g. PS-) diastereoisomers.[11,12]
The MePS2 modi cation in the backbone region adjacent to the 30-dTdT overhang of the siRNA sense strand triggers a strongly enhanced affinity to the RNAinduced silencing complex (RISC) Ago[2] slicer and silencing of proteins that confer chemoresistance in ovarian cancer cell lines and tumors.[15]. This nding is surprising given the fact that the antisense siRNA strand mediates cleavage of the target RNA at the Ago[2] active site and indicates that chemical modi cation of the sense strand can play a key role in siRNA loading and efficacy
We demonstrate here that MePS2 modi cation does not affect RNA conformation and stability
Summary
Unlike the phosphorothioate moiety (PS; Fig. 1), PS2 is achiral, precluding potential drawbacks that may arise from chiral Psubstituted oligonucleotides, owing to variable biophysical and biochemical properties of individual (e.g. PS-) diastereoisomers.[11,12] In addition, PS2-modi ed siRNAs display increased protection against degradation by nucleases compared with PSmodi ed oligonucleotides.[10,13,14] Combining the PS2 with the 20O-methyl ribose modi cation (MePS2-RNA; Fig. 1) afforded increased loading of modi ed siRNA duplexes into the RNAinduced silencing complex (RISC) as well as enhanced antitumor activity.[15] Interestingly, the former property can be attributed to MePS2 modi cation at two residues adjacent to the 30-TT overhang in the sense siRNA strand (ESI Fig. S1†). The 20-O-methyl modi cation can offset the slight thermodynamic destabilization caused by the PS2 moiety compared with the native RNA duplex.[10] We determined the melting temperatures (Tm) of siRNAs containing two to six MePS2 modi cations and found that they exhibit thermal
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