14 research outputs found
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
Asialoglycoprotein receptor 1 mediates productive uptake of N-acetylgalactosamine-conjugated and unconjugated phosphorothioate antisense oligonucleotides into liver hepatocytes
Hsp90 protein interacts with phosphorothioate oligonucleotides containing hydrophobic 2′-modifications and enhances antisense activity
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
Characterizing the effect of GalNAc and phosphorothioate backbone on binding of antisense oligonucleotides to the asialoglycoprotein receptor
Peptide Nucleic Acids Conjugated to Short Basic Peptides Show Improved Pharmacokinetics and Antisense Activity in Adipose Tissue
Dose-dependent reduction of somatic expansions but not Htt aggregates by di-valent siRNA-mediated silencing of MSH3 in HdhQ111 mice
Abstract Huntington's disease (HD) is a progressive neurodegenerative disorder caused by CAG trinucleotide repeat expansions in exon 1 of the HTT gene. In addition to germline CAG expansions, somatic repeat expansions in neurons also contribute to HD pathogenesis. The DNA mismatch repair gene, MSH3, identified as a genetic modifier of HD onset and progression, promotes somatic CAG expansions, and thus presents a potential therapeutic target. However, what extent of MSH3 protein reduction is needed to attenuate somatic CAG expansions and elicit therapeutic benefits in HD disease models is less clear. In our study, we employed potent di-siRNAs to silence mouse Msh3 mRNA expression in a dose-dependent manner in HdhQ111/+ mice and correlated somatic Htt CAG instability with MSH3 protein levels from simultaneously isolated DNA and protein after siRNA treatment. Our results reveal a linear correlation with a proportionality constant of ~ 1 between the prevention of somatic Htt CAG expansions and MSH3 protein expression in vivo, supporting MSH3 as a rate-limiting step in somatic expansions. Intriguingly, despite a 75% reduction in MSH3 protein levels, striatal nuclear HTT aggregates remained unchanged. We also note that evidence for nuclear Msh3 mRNA that is inaccessible to RNA interference was found, and that MSH6 protein in the striatum was upregulated following MSH3 knockdown in HdhQ111/+ mice. These results provide important clues to address critical questions for the development of therapeutic molecules targeting MSH3 as a potential therapeutic target for HD
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