Antisense oligonucleotides capable of promoting specific target mRNA reduction via competing RNase H1-dependent and independent mechanisms.

Timothy A. Vickers,Stanley T. Crooke,Emanuele Buratti

doi:10.1371/journal.pone.0108625

Timothy A. Vickers, Stanley T. Crooke + Show 1 more

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https://doi.org/10.1371/journal.pone.0108625

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Journal: PloS one	Publication Date: Oct 9, 2014
Citations: 59	License type: CC BY 4.0

Affiliation: Ionis Pharmaceuticals (United States)

Abstract

Antisense oligonucleotides (ASOs) are most commonly designed to reduce targeted RNA via RNase H1-dependent degradation. In this paper we demonstrate that cellular proteins can compete for sites targeted by RNase H1-dependent ASOs. We further show that some ASOs designed to mediate RNase H1 cleavage can, in certain instances, promote target reduction both by RNase H1-mediated cleavage and by steric inhibition of binding of splicing factors at a site required for efficient processing of the pre-mRNA. In the latter case, RNase H cleavage was prevented by binding of a second protein, HSPA8, to the ASO/pre-mRNA heteroduplex. In addition, using a precisely controlled minigene system, we directly demonstrated that activity of ASOs targeting sites in introns is strongly influenced by splicing efficiency.

Highlights

Several mechanisms are known by which short synthetic oligonucleotides can be used to modulate gene expression in mammalian cells [1]
We identify DNA-like antisense oligonucleotides (ASOs) that are capable of mediating RNase H1 cleavage and of displacing factors required for efficient splicing of the pre-mRNA
Effects of RNA processing on antisense oligonucleotide activity To evaluate the effects of RNA processing on ASO activity, a minigene system precisely controlled by addition of tetracycline (TET) was utilized [20]

Summary

Introduction

Several mechanisms are known by which short synthetic oligonucleotides can be used to modulate gene expression in mammalian cells [1]. These antisense mechanisms require binding of the oligonucleotide to the targeted RNA and are broadly classified as cleavage-dependent or occupancy-only mechanisms. Human RNase H1 is active as a single peptide, whereas RNase H2 is a heterotrimeric enzyme [3,4] Both enzymes are thought to play a role in DNA replication and repair, but additional biological functions are likely for both. The RNase H1 mechanism has been broadly exploited as both a research tool and a human therapeutic [6]

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