Skip to main content
SpringerLink
Log in

A Direct Comparison of Anti-microRNA Oligonucleotide Potency

  • Research Paper
  • Published:
Pharmaceutical Research Aims and scope Submit manuscript

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

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

REFERENCES

  1. Carthew RW, Sontheimer EJ. Origins and mechanisms of miRNAs and siRNAs. Cell. 2009;136:642–55.

    Article  CAS  PubMed  Google Scholar 

  2. Vaucheret H. Post-transcriptional small RNA pathways in plants: mechanisms and regulations. Genes Dev. 2006;20:759–71.

    Article  CAS  PubMed  Google Scholar 

  3. Lewis BP, Burge CB, Bartel DP. Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell. 2005;120:15–20.

    Article  CAS  PubMed  Google Scholar 

  4. Krek A, Grun D, Poy MN, Wolf R, Rosenberg L, Epstein EJ et al. Combinatorial microRNA target predictions. Nat Genet. 2005;37:495–500.

    Article  CAS  PubMed  Google Scholar 

  5. Alvarez-Garcia I, Miska EA. MicroRNA functions in animal development and human disease. Development. 2005;132:4653–62.

    Article  CAS  PubMed  Google Scholar 

  6. Johnston RJ, Hobert O. A microRNA controlling left/right neuronal asymmetry in Caenorhabditis elegans. Nature. 2003;426:845–9.

    Article  CAS  PubMed  Google Scholar 

  7. Wilfred BR, Wang WX, Nelson PT. Energizing miRNA research: a review of the role of miRNAs in lipid metabolism, with a prediction that miR-103/107 regulates human metabolic pathways. Mol Genet Metab. 2007;91:209–17.

    Article  CAS  PubMed  Google Scholar 

  8. Lindsay MA. microRNAs and the immune response. Trends Immunol. 2008;29:343–51.

    Article  CAS  PubMed  Google Scholar 

  9. Nelson PT, Wang WX, Rajeev BW. MicroRNAs (miRNAs) in neurodegenerative diseases. Brain Pathol. 2008;18:130–8.

    Article  CAS  PubMed  Google Scholar 

  10. Bushati N, Cohen SM. MicroRNAs in neurodegeneration. Curr Opin Neurobiol. 2008;18:292–6.

    Article  CAS  PubMed  Google Scholar 

  11. Thum T, Catalucci D, Bauersachs J. MicroRNAs: novel regulators in cardiac development and disease. Cardiovasc Res. 2008;79:562–70.

    Article  CAS  PubMed  Google Scholar 

  12. Xiao C, Rajewsky K. MicroRNA control in the immune system: basic principles. Cell. 2009;136:26–36.

    Article  CAS  PubMed  Google Scholar 

  13. Esquela-Kerscher A, Slack FJ. Oncomirs—microRNAs with a role in cancer. Nat Rev Cancer. 2006;6:259–69.

    Article  CAS  PubMed  Google Scholar 

  14. Wiemer EA. The role of microRNAs in cancer: no small matter. Eur J Cancer. 2007;43:1529–44.

    Article  CAS  PubMed  Google Scholar 

  15. Croce CM. Causes and consequences of microRNA dysregulation in cancer. Nat Rev Genet. 2009;10:704–14.

    Article  CAS  PubMed  Google Scholar 

  16. Lee Y, Ahn C, Han J, Choi H, Kim J, Yim J et al. The nuclear RNase III Drosha initiates microRNA processing. Nature. 2003;425:415–9.

    Article  CAS  PubMed  Google Scholar 

  17. Lund E, Guttinger S, Calado A, Dahlberg JE, Kutay U. Nuclear export of microRNA precursors. Science. 2004;303:95–8.

    Article  CAS  PubMed  Google Scholar 

  18. Schaefer A, O’Carroll D, Tan CL, Hillman D, Sugimori M, Llinas R et al. Cerebellar neurodegeneration in the absence of microRNAs. J Exp Med. 2007;204:1553–8.

    Article  CAS  PubMed  Google Scholar 

  19. Cai X, Hagedorn CH, Cullen BR. Human microRNAs are processed from capped, polyadenylated transcripts that can also function as mRNAs. RNA. 2004;10:1957–66.

    Article  CAS  PubMed  Google Scholar 

  20. Krutzfeldt J, Rajewsky N, Braich R, Rajeev KG, Tuschl T, Manoharan M et al. Silencing of microRNAs in vivo with ‘antagomirs’. Nature. 2005;438:685–9.

    Article  PubMed  Google Scholar 

  21. Davis S, Lollo B, Freier S, Esau C. Improved targeting of miRNA with antisense oligonucleotides. Nucleic Acids Res. 2006;34:2294–304.

    Article  CAS  PubMed  Google Scholar 

  22. Lennox KA, Sabel JL, Johnson MJ, Moreira BG, Fletcher CA, Rose SD et al. Characterization of modified antisense oligonucleotides in Xenopus laevis embryos. Oligonucleotides. 2006;16:26–42.

    Article  CAS  PubMed  Google Scholar 

  23. Brown DA, Kang SH, Gryaznov SM, DeDionisio L, Heidenreich O, Sullivan S et al. Effect of phosphorothioate modification of oligodeoxynucleotides on specific protein binding. J Biol Chem. 1994;269:26801–5.

    CAS  PubMed  Google Scholar 

  24. Geary RS, Watanabe TA, Truong L, Freier S, Lesnik EA, Sioufi NB et al. Pharmacokinetic properties of 2′-O-(2-methoxyethyl)-modified oligonucleotide analogs in rats. J Pharmacol Exp Ther. 2001;296:890–7.

    CAS  PubMed  Google Scholar 

  25. Freier SM, Altmann KH. The ups and downs of nucleic acid duplex stability: structure-stability studies on chemically-modified DNA:RNA duplexes. Nucleic Acids Res. 1997;25:4429–43.

    Article  CAS  PubMed  Google Scholar 

  26. Petersen M, Bondensgaard K, Wengel J, Jacobsen JP. Locked nucleic acid (LNA) recognition of RNA: NMR solution structures of LNA:RNA hybrids. J Am Chem Soc. 2002;124:5974–82.

    Article  CAS  PubMed  Google Scholar 

  27. Kurreck J, Wyszko E, Gillen C, Erdmann VA. Design of antisense oligonucleotides stabilized by locked nucleic acids. Nucleic Acids Res. 2002;30:1911–8.

    Article  CAS  PubMed  Google Scholar 

  28. Crinelli R, Bianchi M, Gentilini L, Magnani M. Design and characterization of decoy oligonucleotides containing locked nucleic acids. Nucleic Acids Res. 2002;30:2435–43.

    Article  CAS  PubMed  Google Scholar 

  29. Frieden M, Hansen HF, Koch T. Nuclease stability of LNA oligonucleotides and LNA-DNA chimeras. Nucleos Nucleot Nucleic Acids. 2003;22:1041–3.

    Article  CAS  Google Scholar 

  30. Petersen M, Wengel J. LNA: a versatile tool for therapeutics and genomics. Trends Biotechnol. 2003;21:74–81.

    Article  CAS  PubMed  Google Scholar 

  31. Cheng AM, Byrom MW, Shelton J, Ford LP. Antisense inhibition of human miRNAs and indications for an involvement of miRNA in cell growth and apoptosis. Nucleic Acids Res. 2005;33:1290–7.

    Article  CAS  PubMed  Google Scholar 

  32. Hutvagner G, Simard MJ, Mello CC, Zamore PD. Sequence-specific inhibition of small RNA function. PLoS Biol. 2004;2:E98.

    Article  PubMed  Google Scholar 

  33. Meister G, Landthaler M, Dorsett Y, Tuschl T. Sequence-specific inhibition of microRNA- and siRNA-induced RNA silencing. Rna. 2004;10:544–50.

    Article  CAS  PubMed  Google Scholar 

  34. Orom UA, Kauppinen S, Lund AH. LNA-modified oligonucleotides mediate specific inhibition of microRNA function. Gene. 2006;372:137–41.

    Article  CAS  PubMed  Google Scholar 

  35. Vermeulen A, Robertson B, Dalby AB, Marshall WS, Karpilow J, Leake D et al. Double-stranded regions are essential design components of potent inhibitors of RISC function. Rna. 2007;13:723–30.

    Article  CAS  PubMed  Google Scholar 

  36. Esau C, Davis S, Murray SF, Yu XX, Pandey SK, Pear M et al. miR-122 regulation of lipid metabolism revealed by in vivo antisense targeting. Cell Metab. 2006;3:87–98.

    Article  CAS  PubMed  Google Scholar 

  37. Kloosterman WP, Lagendijk AK, Ketting RF, Moulton JD, Plasterk RH. Targeted inhibition of miRNA maturation with morpholinos reveals a role for miR-375 in pancreatic islet development. PLoS Biol. 2007;5:e203.

    Article  PubMed  Google Scholar 

  38. Leaman D, Chen PY, Fak J, Yalcin A, Pearce M, Unnerstall U et al. Antisense-mediated depletion reveals essential and specific functions of microRNAs in Drosophila development. Cell. 2005;121:1097–108.

    Article  CAS  PubMed  Google Scholar 

  39. Elmen J, Lindow M, Silahtaroglu A, Bak M, Christensen M, Lind-Thomsen A et al. Antagonism of microRNA-122 in mice by systemically administered LNA-antimiR leads to up-regulation of a large set of predicted target mRNAs in the liver. Nucleic Acids Res. 2008;36:1153–62.

    Article  CAS  PubMed  Google Scholar 

  40. Elmen J, Lindow M, Schutz S, Lawrence M, Petri A, Obad S et al. LNA-mediated microRNA silencing in non-human primates. Nature. 2008;452:896–9.

    Article  CAS  PubMed  Google Scholar 

  41. Davis S, Propp S, Freier SM, Jones LE, Serra MJ, Kinberger G et al. Potent inhibition of microRNA in vivo without degradation. Nucleic Acids Res. 2009;37:70–7.

    Article  CAS  PubMed  Google Scholar 

  42. Kurreck J. Antisense technologies. Improvement through novel chemical modifications. Eur J Biochem. 2003;270:1628–44.

    Article  CAS  PubMed  Google Scholar 

  43. Eder PS, DeVine RJ, Dagle JM, Walder JA. Substrate specificity and kinetics of degradation of antisense oligonucleotides by a 3′ exonuclease in plasma. Antisense Res Dev. 1991;1:141–51.

    CAS  PubMed  Google Scholar 

  44. Dagle JM, Weeks DL, Walder JA. Pathways of degradation and mechanism of action of antisense oligonucleotides in Xenopus laevis embryos. Antisense Res Dev. 1991;1:11–20.

    CAS  PubMed  Google Scholar 

  45. Koziolkiewicz M, Wojcik M, Kobylanska A, Karwowski B, Rebowska B, Guga P et al. Stability of stereoregular oligo(nucleoside phosphorothioate)s in human plasma: diastereoselectivity of plasma 3′-exonuclease. Antisense Nucleic Acid Drug Dev. 1997;7:43–8.

    CAS  PubMed  Google Scholar 

  46. Gilar M, Belenky A, Budman Y, Smisek DL, Cohen AS. Impact of 3′-exonuclease stereoselectivity on the kinetics of phosphorothioate oligonucleotide metabolism. Antisense Nucleic Acid Drug Dev. 1998;8:35–42.

    CAS  PubMed  Google Scholar 

  47. Krutzfeldt J, Kuwajima S, Braich R, Rajeev KG, Pena J, Tuschl T et al. Specificity, duplex degradation and subcellular localization of antagomirs. Nucleic Acids Res. 2007;35:2885–92.

    Article  CAS  PubMed  Google Scholar 

  48. Tang JY, Temsamani J, Agrawal S. Self-stabilized antisense oligodeoxynucleotide phosphorothioates: properties and anti-HIV activity. Nucleic Acids Res. 1993;21:2729–35.

    Article  CAS  PubMed  Google Scholar 

  49. Hosono K, Kuwasaki T, Tsukahara S, Takai K, Takaku H. Properties of base-pairing in the stem region of hairpin antisense oligonucleotides containing 2′-methoxynucleosides. Biochim Biophys Acta. 1995;1244:339–44.

    CAS  PubMed  Google Scholar 

  50. Lanford RE, Hildebrandt-Eriksen ES, Petri A, Persson R, Lindow M, Munk ME et al. Therapeutic silencing of microRNA-122 in primates with chronic hepatitis C virus infection. Science. 2010;327:198–201.

    Article  CAS  PubMed  Google Scholar 

  51. Chan JA, Krichevsky AM, Kosik KS. MicroRNA-21 is an antiapoptotic factor in human glioblastoma cells. Cancer Res. 2005;65:6029–33.

    Article  CAS  PubMed  Google Scholar 

  52. Weiler J, Hunziker J, Hall J. Anti-miRNA oligonucleotides (AMOs): ammunition to target miRNAs implicated in human disease? Gene Ther. 2006;13:496–502.

    Article  CAS  PubMed  Google Scholar 

  53. You Y, Moreira BG, Behlke MA, Owczarzy R. Design of LNA probes that improve mismatch discrimination. Nucleic Acids Res. 2006;34:e60.

    Article  PubMed  Google Scholar 

  54. Bartel DP. MicroRNAs: target recognition and regulatory functions. Cell. 2009;136:215–33.

    Article  CAS  PubMed  Google Scholar 

Download references

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.

Author information

Authors and Affiliations

  1. Integrated DNA Technologies, 1710 Commercial Park, Coralville, Iowa, 52241, USA

    Kim A. Lennox & Mark A. Behlke

Authors
  1. Kim A. Lennox
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Mark A. Behlke
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Mark A. Behlke.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Table S1

Anti-miRNA Oligonucleotides (PDF 15 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)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Lennox, K.A., Behlke, M.A. A Direct Comparison of Anti-microRNA Oligonucleotide Potency. Pharm Res 27, 1788–1799 (2010). https://doi.org/10.1007/s11095-010-0156-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11095-010-0156-0

KEY WORDS

Search

Navigation