2 research outputs found
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