Potent inhibition of microRNA in vivo without degradation

Chemically modified antisense oligonucleotides (ASOs) are widely used as a tool to functionalize microRNAs (miRNAs). Reduction of miRNA level after ASO inhibition is commonly reported to show efficacy. Whether this is the most relevant endpoint for measuring miRNA inhibition has not been adequately addressed in the field although it has important implications for evaluating miRNA targeting studies. Using a novel approach to quantitate miRNA levels in the presence of excess ASO, we have discovered that the outcome of miRNA inhibition can vary depending on the chemical modification of the ASO. Although some miRNA inhibitors cause a decrease in mature miRNA levels, we have identified a novel 2′-fluoro/2′-methoxyethyl modified ASO motif with dramatically improved in vivo potency which does not. These studies show there are multiple mechanisms of miRNA inhibition by ASOs and that evaluation of secondary endpoints is crucial for interpreting miRNA inhibition studies

S-Acyl-2-Thioethyl: A Convenient Base-Labile Protecting Group for the Synthesis of siRNAs Containing 5′-Vinylphosphonate

We recently reported that (E)-5′-vinylphosphonate (5′-VP) is a metabolically-stable phosphate mimic for siRNA and demonstrated that 5′-VP improves the potency of the fully modified siRNAs in vivo. Here, we report an alternative synthesis of 5′-VP modified guide strand using S-pivaloyl-2-thioethyl (tBu-SATE) protecting group. The tBu-SATE group is readily removed during the final cleavage of the oligonucleotide from the solid support and providing a more convenient route for the synthesis of siRNA guide strand carrying a 5′-vinylphosphonate

Structural Analysis of Human Argonaute‑2 Bound to a Modified siRNA Guide

Incorporation of chemical modifications into small interfering RNAs (siRNAs) increases their metabolic stability and improves their tissue distribution. However, how these modifications impact interactions with Argonaute-2 (Ago2), the molecular target of siRNAs, is not known. Herein we present the crystal structure of human Ago2 bound to a metabolically stable siRNA containing extensive backbone modifications. Comparison to the structure of an equivalent unmodified-siRNA complex indicates that the structure of Ago2 is relatively unaffected by chemical modifications in the bound siRNA. In contrast, the modified siRNA appears to be much more plastic and shifts, relative to the unmodified siRNA, to optimize contacts with Ago2. Structure–activity analysis reveals that even major conformational perturbations in the 3′ half of the siRNA seed region have a relatively modest effect on knockdown potency. These findings provide an explanation for a variety of modification patterns tolerated in siRNAs and a structural basis for advancing therapeutic siRNA design

Efficient Synthesis and Biological Evaluation of 5′-GalNAc Conjugated Antisense Oligonucleotides

Conjugation of triantennary <i>N</i>-acetyl galactosamine (GalNAc) to oligonucleotide therapeutics results in marked improvement in potency for reducing gene targets expressed in hepatocytes. In this report we describe a robust and efficient solution-phase conjugation strategy to attach triantennary GalNAc clusters (mol. wt. ∼2000) activated as PFP (pentafluorophenyl) esters onto 5′-hexylamino modified antisense oligonucleotides (5′-HA ASOs, mol. wt. ∼8000 Da). The conjugation reaction is efficient and was used to prepare GalNAc conjugated ASOs from milligram to multigram scale. The solution phase method avoids loading of GalNAc clusters onto solid-support for automated synthesis and will facilitate evaluation of GalNAc clusters for structure activity relationship (SAR) studies. Furthermore, we show that transfer of the GalNAc cluster from the 3′-end of an ASO to the 5′-end results in improved potency in cells and animals