Expanding Conjugate Space of RNAi Therapeutics: Ligand at the 3′ End of the Antisense Strand Achieves Uncompromised In Vivo Potency and Efficacy and Reveals Interactions with the Argonaute-2 PAZ Domain

-acetylgalactosamine (GalNAc), the ligand for a hepatocyte-specific receptor, enables the delivery of multiple clinically approved therapeutic agents that act through the RNA interference pathway. Here, we report the systematic evaluation of siRNAs with the 3' termini of antisense strands conjugated to GalNAc for the first time. These designs retained the same receptor affinity, in vitro and in vivo activities, as well as the same level of loading into the RNA-induced silencing complex as siRNAs with a GalNAc-conjugated sense strand. A siRNA with a GalNAc-conjugated antisense strand of 22 nucleotides had better activity than a siRNA with a 23-nucleotide antisense strand. Computational modeling of a complex of a GalNAc-conjugated antisense strand with the PAZ domain of Ago2 rationalizes the importance of the interaction of phosphate at the 3' terminus with the PAZ domain to explain the observed activity of these siRNAs.