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Pharmaceutical Research

, Volume 27, Issue 9, pp 1788–1799 | Cite as

A Direct Comparison of Anti-microRNA Oligonucleotide Potency

  • Kim A. Lennox
  • Mark A. BehlkeEmail author
Research Paper

ABSTRACT

Purpose

Cataloguing endogenous miRNA targets by inhibiting miRNA function is fundamental to understanding the biological importance of each miRNA in gene regulatory pathways. Methods to down-regulate miRNA activity may help treat diseases where over-expression of miRNAs relates to the underlying pathophysiology. This study objectively evaluates the in vitro potency of different anti-miRNA oligonucleotides (AMOs) using various design and modification strategies described in the literature as well as some novel modification strategies.

Methods

MiR21 and miR16 AMOs, containing chemical modifications such as 2′-O-methyl RNA, locked nucleic acid and 2′-Fluoro bases with or without phosphorothioate linkages, were directly compared by transfection into HeLa cells using a dual-luciferase reporter assay to quantify miRNA inhibition.

Results

Potency for the various AMOs ranged from inactive at high dose (50 nM) to strongly inhibitory at both high and low dose (1 nM). Including phosphorothioate linkages improved nuclease stability and generally increased functional potency.

Conclusions

Incorporating high binding affinity modifications, such as LNA and 2′F bases, increases AMO potency while maintaining specificity; nevertheless, use of low dose is preferred when using high potency reagents to minimize the potential for cross reactivity. 2′OMe/LNA chimeras with PS modifications were the most potent constructs tested for miRNA inhibition in vitro.

KEY WORDS

antagonist antisense LNA miRNA potency 

ABBREVIATIONS

2′F

2′-F RNA

2′OMe

2′-O-methyl RNA

AMO

anti-miRNA oligonucleotide

ASO

antisense oligonucleotide

LNA

locked nucleic acids

miRNA

microRNA

RISC

RNA induced silencing complex

RNAi

RNA interference

Tm

melting temperature

UTR

untranslated region

Notes

ACKNOWLEDGEMENTS

We thank Dr. Scott Rose for making the mouse liver protein extracts used in the nuclease stability studies. This research was entirely supported by internal funding from Integrated DNA Technologies, Inc.

Supplementary material

11095_2010_156_MOESM1_ESM.pdf (15 kb)
Table S1 Anti-miRNA Oligonucleotides (PDF 15 kb)
11095_2010_156_MOESM2_ESM.pdf (483 kb)
Fig. S1 Effect of chemical modifications on the stability of oligonucleotides in mouse liver protein extract. A) AMOs were designed using DNA, 2′-O-methyl RNA (2′OMe), locked nucleic acid (LNA) and 2′-Fluoro (2′F) bases with varying degrees of phosphorothioate (PS) linkages. B) 8 μM of each AMO was incubated in 20% mouse liver protein extract for 0, 2, 6 or 24 hrs, with the reactions stopped by adding equal volumes to 2X formamide gel loading buffer, flash freezing on dry ice and storage at −80°C. 40 pmoles of each AMO was separated on 14% polyacrylamide gels supplemented with 8M urea and 20% formamide, stained with 1X GelStar, and visualized using UV excitation. (PDF 482 kb)

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Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  1. 1.Integrated DNA TechnologiesCoralvilleUSA

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