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Quantification of Oligonucleotide Association with miRNA–Argonaute Complexes In Vitro

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Drug Target miRNA

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1517))

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Abstract

A major challenge in the development of oligonucleotide-based microRNA (miRNA) inhibitors for therapeutic applications is the identification of candidate designs with strong affinity for the target miRNA in the context of the Argonaute complex. To this effect, distinct chemical modifications are employed along the length of the oligonucleotide aimed at strengthening the interactions with the target miRNA. However, the modification chemistry and placement can inadvertently affect the intrinsic ability of the oligonucleotide to pair with its target in the context of Argonaute. To facilitate the design of potent oligonucleotides, we developed a sensitive high-throughput methodology to compare anti-miR compounds for their ability to associate with the miRNA/Argonaute complex.

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References

  1. Mendell JT, Olson EN (2012) MicroRNAs in stress signaling and human disease. Cell 148:1172–1187

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Seyhan AA (2015) microRNAs with different functions and roles in disease development and as potential biomarkers of diabetes: progress and challenges. Mol Biosyst 11:1217–1234

    Article  CAS  PubMed  Google Scholar 

  3. Chi SW, Zang JB, Mele A, Darnell RB (2009) Argonaute HITS-CLIP decodes microRNA-mRNA interaction maps. Nature 460:479–486

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Zisoulis DG, Lovci MT, Wilbert ML, Hutt KR, Liang TY et al (2010) Comprehensive discovery of endogenous Argonaute binding sites in Caenorhabditis elegans. Nature structural & molecular biology 17:173–179

    Article  CAS  Google Scholar 

  5. Hutvagner G, Simard MJ, Mello CC, Zamore PD (2004) Sequence-specific inhibition of small RNA function. PLoS biology 2, E98

    Article  PubMed  PubMed Central  Google Scholar 

  6. Meister G, Landthaler M, Dorsett Y, Tuschl T (2004) Sequence-specific inhibition of microRNA- and siRNA-induced RNA silencing. RNA 10:544–550

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Jopling CL, Yi M, Lancaster AM, Lemon SM, Sarnow P (2005) Modulation of hepatitis C virus RNA abundance by a liver-specific MicroRNA. Science 309:1577–1581

    Article  CAS  PubMed  Google Scholar 

  8. Krützfeldt J, Rajewsky N, Braich R, Rajeev KG, Tuschl T et al (2005) Silencing of microRNAs in vivo with ‘antagomirs’. Nature 438:685–689

    Article  PubMed  Google Scholar 

  9. Davis S, Lollo B, Freier S, Esau C (2006) Improved targeting of miRNA with antisense oligonucleotides. Nucleic acids research 34:2294–2304

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  11. Janssen HL, Kauppinen S, Hodges MR (2013) HCV infection and miravirsen. N Engl J Med 369:878

    CAS  PubMed  Google Scholar 

  12. Janssen HL, Reesink HW, Lawitz EJ, Zeuzem S, Rodriguez-Torres M et al (2013) Treatment of HCV infection by targeting microRNA. N Engl J Med 368:1685–1694

    Article  CAS  PubMed  Google Scholar 

  13. Van Der Ree MLV, Stelma F, Willemse S, van der Valk M, Rietdijk S, Molenkamp R, Schinkel J et al (2015) A single subcutaneous dose of 2 mg/kg or 4 mg/kg of RG-101, a GalNAc-conjugated oligonucleotide with antagonist activity against MIR-122, results in significant viral load reductions in chronic hepatitis C patients. Journal of Hepatology 62:261

    Article  Google Scholar 

  14. Bartel DP (2009) MicroRNAs: target recognition and regulatory functions. Cell 136:215–233

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Breda J, Rzepiela AJ, Gumienny R, van Nimwegen E, Zavolan M (2015) Quantifying the strength of miRNA-target interactions. Methods 85:90–9

    Article  CAS  PubMed  Google Scholar 

  16. Hammell M, Long D, Zhang L, Lee A, Carmack CS et al (2008) mirWIP: microRNA target prediction based on microRNA-containing ribonucleoprotein-enriched transcripts. Nat Methods 5:813–819

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Esau CC (2008) Inhibition of microRNA with antisense oligonucleotides. Methods 44:55–60

    Article  CAS  PubMed  Google Scholar 

  18. Stenvang J, Petri A, Lindow M, Obad S, Kauppinen S (2012) Inhibition of microRNA function by antimiR oligonucleotides. Silence 3:1

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Crooke ST (2008) Antisense drug technology: principles, strategies, and applications. CRC Press, Boca Raton, FL, xvii, 825 p, 812 p. of plates p

    Google Scholar 

  20. Hogan DJ, Vincent TM, Fish S, Marcusson EG, Bhat B et al (2014) Anti-miRs competitively inhibit microRNAs in Argonaute complexes. PLoS One 9, e100951

    Article  PubMed  PubMed Central  Google Scholar 

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Correspondence to Dimitrios G. Zisoulis .

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Zisoulis, D.G. (2017). Quantification of Oligonucleotide Association with miRNA–Argonaute Complexes In Vitro. In: Schmidt, M. (eds) Drug Target miRNA. Methods in Molecular Biology, vol 1517. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6563-2_4

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  • DOI: https://doi.org/10.1007/978-1-4939-6563-2_4

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6561-8

  • Online ISBN: 978-1-4939-6563-2

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