siRNAs are short dsRNAs that mediate efficient target gene silencing in a sequence-specific manner. We previously developed a novel siRNA structure, called asiRNA (asymmetric siRNA), which alleviates the off-target effects associated with conventional siRNA structures without decreasing target gene silencing potency. In the present study, we explored the effect of the guide strand 3'-end structure on the gene silencing potency of asiRNA. Interestingly, asiRNAs with a 21 nt guide strand solely composed of RNA resulted in gene silencing that was more than 6-fold more efficient compared with the corresponding asiRNA guide strand harbouring a dTdT (deoxythymidine dinucleotide) at its 3'-end. We demonstrated that the molecular basis of potency of the asiRNA with a 21 nt guide strand composed solely of RNA was due to the enhanced formation of the RISC (RNA-induced silencing complex) and increased affinity towards hAgo2 (human Argonaute2). Our observations may assist researchers in designing new asiRNAs with high on-target silencing efficiency with low off-target effects, which is critical for applications in both basic research and therapeutic development.