To assess whether SIDT2 is able to interact with siRNA, we adapted a method combining Fluorescence Lifetime Imaging (FLIM) with Förster Resonance Energy Transfer (FRET) used in a previous study to assess the ability of SIDT2 to interact with long dsRNA in the form of poly(I:C). Unlike long dsRNA, we found that naked siRNAs showed a poor cellular uptake. To address this, we complexed fluorescein-conjugated siRNAs with a transfection reagent to facilitate endocytosis prior to the treatment of mouse embryonic fibroblasts (MEFs) that stably expressed SIDT2-mCherry. Using this approach, we observed co-localization of fluorescein-conjugated siRNAs with SIDT2-mCherry, consistent with efficient internalization via endocytosis (Fig. 1A). We subsequently performed FLIM-FRET analysis 16 h post-treatment. Notably, we observed a significant reduction in fluorescence lifetime for siRNA-fluorescein in the presence of SIDT2-mCherry (Fig. 1B) indicating a likely molecular interaction between SIDT2 and siRNA. To test whether this interaction was specific to siRNA, we performed the same experiment using fluorescein-conjugated dsDNA and consistent with the previous reports, we did not observe a reduction in fluorescence lifetime dsDNA-fluorescein and SIDT2-mCherry (Fig. 1C), suggesting that SIDT2 can distinguish between siRNA and dsDNA as has previously been shown for C. elegans SID-1.
Although unmodified siRNAs work well to silence gene activity in vitro, siRNAs are usually modified prior to delivery in vivo to counteract ribonuclease degradation, immunogenicity, off-target effects and unfavorable pharmacokinetics. The types of modifications that have been utilized are extensive, and include chemical changes to the ribose sugar, phosphodiester backbone, and nitrogenous bases (Fig. 1D). How such modifications affect transport by SIDT2 is currently unknown, but RNA transport by C. elegans SID-1 has been shown to be impaired when modifications are made to the ribose sugar.
Structural analysis of SID-1 and it's mammalian orthologs suggest that these proteins likely function as multimeric transmembrane channels that transport dsRNA via a central pore (Fig. 1E). Given this, we hypothesized that while unmodified siRNAs can thread this pore, bulky chemical modifications to the ribose sugar or phosphodiester backbone will sterically hinder transit through the pore (Fig. 1F-G). To test this, we again used FLIM-FRET to determine whether common siRNA modifications previously used in clinical trials affect SIDT2 interaction. Notably, we find that bulky modifications such as phosphothiorate (PS) and 2′-O-methyl (2′-OMe) substitutions result in loss of interaction with SIDT2, while more compact modifications such as 2′-O-fluoro (2′-F) substitution maintain the interaction (Fig. 1H). This is consistent with our hypothesis regarding steric hindrance and provides evidence that bulky siRNA modifications are detrimental to SIDT2 interaction. Moreover, these results might help to also explain why gene knockdown is less efficient when PS and 2′-OMe groups are added to unmodified siRNAs.