We report on the synthesis of siRNAs containing both 2′-5′- and 3′-5′-internucleotide linkages and their effects on siRNA structure, function, and interaction with RNAi proteins. Screening of these siRNAs against their corresponding mRNA targets showed that 2′-5′ linkages were well tolerated in the sense strand, but only at a few positions in the antisense strand. Extensive modification of the antisense strand minimally affected 5′-phosphorylation of the siRNA by kinases, however, it negatively affected siRNA loading into human AGO2. Modelling and molecular dynamics simulations were fully consistent with these findings. Furthermore, our studies indicated that the presence of a single 5′p-rN1-(2′-5′)-N2 unit in the antisense strand does not alter the ‘clover leaf’ bend and sugar puckers that are critical for anchoring the 5′-phosphate to Ago 2 MID domain. Importantly, 2′-5′-linkages had the added benefit of abrogating immune-stimulatory activity of siRNAs. Together, these results demonstrate that 2′-5′/3′-5′-modified siRNAs, when properly designed, can offer an efficient new class of siRNAs with diminished immune-stimulatory responses.