Improved Performance of Anti-miRNA Oligonucleotides Using a Novel Non-Nucleotide Modifier

Kim A Lennox,Richard Owczarzy,Mark A Behlke,Joseph A Walder,Derek M Thomas

doi:10.1038/mtna.2013.46

Kim A Lennox, Richard Owczarzy + Show 3 more

Open Access

https://doi.org/10.1038/mtna.2013.46

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Abstract

Anti-microRNA oligonucleotides (AMOs) are steric blocking antisense reagents that inhibit microRNA (miRNA) function by hybridizing and repressing the activity of a mature miRNA. First generation AMOs employed 2′-O-Methyl RNA nucleotides (2′OMe) with phosphorothioate (PS) internucleotide linkages positioned at both ends to block exonuclease attack. Second generation AMOs improved potency through the use of chemical modifications that increase binding affinity to the target, such as locked nucleic acid (LNA) residues. However, this strategy can reduce specificity as high binding affinity compounds can bind to and suppress function of related sequences even if one or more mismatches are present. Further, unnatural modified nucleic acid residues can have toxic side effects. In the present study, a variety of non-nucleotide modifiers were screened for utility in steric blocking antisense applications. A novel compound, N,N-diethyl-4-(4-nitronaphthalen-1-ylazo)-phenylamine (“ZEN”), was discovered that increased binding affinity and blocked exonuclease degradation when placed at or near each end of a single-stranded oligonucleotide. This new modification was combined with the 2′OMe RNA backbone to make ZEN-AMOs. The new ZEN-AMOs have high potency and can effectively inhibit miRNA function in vitro at low nanomolar concentrations, show high specificity, and have low toxicity in cell culture.

Highlights

MicroRNAs are important small noncoding RNAs responsible for spatiotemporally fine tuning gene expression at the post-transcriptional level
anti-miRNA oligonucleotide (AMO) are a version of steric blocking antisense oligonucleotides that form a duplex with the miRNA guide strand and this binding event is the basis for miRNA inactivation
Inactivation requires that binding affinity between the miRNA and the AMO is significantly higher than that of the natural miRNA guide strand/passenger strand interaction; inhibition of miRNA activity requires that the AMO can hybridize to the miRNA guide strand whether it exists in single-stranded form, double-stranded form with the natural passenger strand, or bound to an Argonaute protein in the miRNA induced silencing complex

Summary

Introduction

MicroRNAs (miRNAs) are important small noncoding RNAs responsible for spatiotemporally fine tuning gene expression at the post-transcriptional level. 20–24 nucleotides long, these small RNA molecules regulate gene expression through imperfect base pairing in the 3′UTR of targeted genes, leading to mRNA degradation or preventing protein synthesis.[1] Just about every cellular biological process, including cell differentiation, apoptosis, and proliferation, is controlled by the miRNA regulatory network.[2,3] The wide acknowledgement of miRNAs as effective biological regulators has amplified the need for techniques to overexpress or inhibit their function both to investigate function as well as for therapeutic purposes to correct diseases associated with miRNA dysregulation.[4]. It has long been appreciated that chemical modifications which increase duplex stability usually increase the potency of antisense oligonucleotides, whether used in steric blocking[5,6,7] or RNase H degradative modes of action.[8,9] The binding affinity of an AMO must be sufficiently high that it cannot be removed from the miRNA target by helicase activities present in miRISC or by competition from other natural targets within the cell. Several AMO designs which satisfy most of these criteria have been developed and have been used with good results to inhibit miRNA function both in vitro and in vivo.[4,6,7]

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