||Despite the tremendous potential of short interfering RNA (siRNA) as a novel biopharmaceutical, its therapeutic utility has not been maximized mainly due to lack of proper in vivo delivery vehicle, off-target effects and several off-pathway protein interactions instigating immunostimulation. Judicious chemical modification of different parts of the siRNA was foreseen as a potential solution to the off-target gene silencing and the off-pathway protein binding. In this study, 8-alkoxyadenosines were explored as a nucleobase modification in the context of the siRNA-based RNA interference (RNAi). These nucleosides are unusual in that they have the potential to exist as an equilibrium mixture of the syn and anti conformers. When placed opposite to U in the complementary strand, 8- alkoxyadenosines can exist in normal anti conformation and form canonical Watson- Crick hydrogen bonding; interestingly, with G as the base-pairing partner, these unusual nucleosides can potentially flip into the syn conformation and form unorthodox Hoogsteen base-pairing. 8-Alkoxyadenosine phosphoramidites were synthesized and incorporated into the guide strand of caspase 2 siRNA at four different positions - two in the seed region, one at the cleavage junction and another nearer to the 3´-end of the guide strands. Thermal stabilities of the corresponding siRNA duplexes showed that U is still preferred over G as the base-pairing partner in the complementary strand. When compared to the unmodified iv positive control siRNAs, singly modified siRNAs have knocked down caspase 2 insert mRNA (generated from a recombinant plasmid) efficiently and with little or no loss of efficacy. Doubly modified siRNAs were found to be less effective and lose their efficacy at low nanomolar concentrations. Persistent placement of steric blockade in the minor groove affected the RNAi efficacy significantly; this observation supports the hypothesis and indicates the necessity of ‘switching' the bulky alkyloxy groups in the major groove, when modified siRNAs interact with the RISC assembly. SiRNAs modified at positions 6 and 10 of the guide strand were found to be effective against preventing interaction with the RNA-dependent protein kinase (PKR). In summary, 8-alkoxyadenosine-containing siRNAs prevented undesired off-pathway protein binding, without compromising the RNAi efficacy significantly.