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Knockdown of Nuclear-Retained Long Noncoding RNAs Using Modified DNA Antisense Oligonucleotides

  • Xinying Zong
  • Lulu Huang
  • Vidisha Tripathi
  • Raechel Peralta
  • Susan M. Freier
  • Shuling Guo
  • Kannanganattu V. Prasanth
Part of the Methods in Molecular Biology book series (MIMB, volume 1262)

Abstract

Long noncoding RNAs (lncRNAs) have recently emerged as important players in diverse cellular processes. Among them, a large fraction of lncRNAs are localized within cell nucleus. And several of these nuclear-retained lncRNAs have been found to regulate key nuclear processes, which brings up the requirement of effective genetic tools to explore the functions of this “dark matter” inside the nucleus. While siRNAs and shRNAs are widely used tools in loss-of-function studies, their general efficiency in depleting nuclear-retained lncRNAs is limited, due to the fact that the RNAi machinery is located mainly in the cytoplasm of mammalian cells. Here, we describe the usage of chemically modified chimeric DNA antisense oligonucleotides (ASO) in effective knockdown of nuclear-retained lncRNAs, with a focus on the detailed workflow from the design and synthesis of ASOs, to in vitro and in vivo delivery methods.

Key words

Chemically modified chimeric DNA antisense oligonucleotides (ASO) Long noncoding RNAs (lncRNAs) Nuclear-retained long noncoding RNAs (nr-lncRNAs) Knockdown Lipid transfection Electroporation Free uptake In vivo delivery 

Notes

Acknowledgements

Research in the KVP lab is supported by grants from NIH/NIGMS (GM088252) and American Cancer Society (RSG-11-174-01-RMC).

References

  1. 1.
    Batista PJ, Chang HY (2013) Long noncoding RNAs: cellular address codes in development and disease. Cell 152(6):1298–1307PubMedCentralPubMedCrossRefGoogle Scholar
  2. 2.
    Nagano T, Fraser P (2011) No-nonsense functions for long noncoding RNAs. Cell 145(2):178–181PubMedCrossRefGoogle Scholar
  3. 3.
    Rinn JL, Chang HY (2012) Genome regulation by long noncoding RNAs. Annu Rev Biochem 81:145–166PubMedCrossRefGoogle Scholar
  4. 4.
    Singh DK, Prasanth KV (2013) Functional insights into the role of nuclear-retained long noncoding RNAs in gene expression control in mammalian cells. Chromosome Res 21(6–7):695–711PubMedCentralPubMedCrossRefGoogle Scholar
  5. 5.
    Nakagawa S, Kageyama Y (2014) Nuclear lncRNAs as epigenetic regulators-Beyond skepticism. Biochim Biophys Acta 1839(3):215–222Google Scholar
  6. 6.
    Tripathi V et al (2010) The nuclear-retained noncoding RNA MALAT1 regulates alternative splicing by modulating SR splicing factor phosphorylation. Mol Cell 39(6):925–938PubMedCentralPubMedCrossRefGoogle Scholar
  7. 7.
    Tripathi V et al (2013) Long noncoding RNA MALAT1 controls cell cycle progression by regulating the expression of oncogenic transcription factor B-MYB. PLoS Genet 9(3):e1003368PubMedCentralPubMedCrossRefGoogle Scholar
  8. 8.
    Gagnon KT, Li L, Chu Y, Janowski BA, Corey DR (2014) RNAi factors are present and active in human cell nuclei. Cell Rep 6(1):211–221PubMedCrossRefGoogle Scholar
  9. 9.
    Billy E, Brondani V, Zhang H, Muller U, Filipowicz W (2001) Specific interference with gene expression induced by long, double-stranded RNA in mouse embryonal teratocarcinoma cell lines. Proc Natl Acad Sci U S A 98(25):14428–14433PubMedCentralPubMedCrossRefGoogle Scholar
  10. 10.
    Zeng Y, Cullen BR (2002) RNA interference in human cells is restricted to the cytoplasm. RNA 8(7):855–860PubMedCentralPubMedCrossRefGoogle Scholar
  11. 11.
    Kawasaki H, Taira K (2003) Short hairpin type of dsRNAs that are controlled by tRNA(Val) promoter significantly induce RNAi-mediated gene silencing in the cytoplasm of human cells. Nucleic Acids Res 31(2):700–707PubMedCentralPubMedCrossRefGoogle Scholar
  12. 12.
    Chiu YL, Ali A, Chu CY, Cao H, Rana TM (2004) Visualizing a correlation between siRNA localization, cellular uptake, and RNAi in living cells. Chem Biol 11(8):1165–1175PubMedCrossRefGoogle Scholar
  13. 13.
    Crooke ST (1999) Molecular mechanisms of action of antisense drugs. Biochim Biophys Acta 1489(1):31–44PubMedCrossRefGoogle Scholar
  14. 14.
    Vickers TA et al (2003) Efficient reduction of target RNAs by small interfering RNA and RNase H-dependent antisense agents. A comparative analysis. J Biol Chem 278(9):7108–7118PubMedCrossRefGoogle Scholar
  15. 15.
    Ideue T, Hino K, Kitao S, Yokoi T, Hirose T (2009) Efficient oligonucleotide-mediated degradation of nuclear noncoding RNAs in mammalian cultured cells. RNA 15(8):1578–1587PubMedCentralPubMedCrossRefGoogle Scholar
  16. 16.
    Lima WF et al (2007) Human RNase H1 discriminates between subtle variations in the structure of the heteroduplex substrate. Mol Pharmacol 71(1):83–91PubMedCrossRefGoogle Scholar
  17. 17.
    Koller E et al (2011) Mechanisms of single-stranded phosphorothioate modified antisense oligonucleotide accumulation in hepatocytes. Nucleic Acids Res 39(11):4795–4807PubMedCentralPubMedCrossRefGoogle Scholar
  18. 18.
    Zhang B et al (2012) The lncRNA Malat1 is dispensable for mouse development but its transcription plays a cis-regulatory role in the adult. Cell Rep 2(1):111–123PubMedCentralPubMedCrossRefGoogle Scholar
  19. 19.
    Graham MJ et al (2007) Antisense inhibition of proprotein convertase subtilisin/kexin type 9 reduces serum LDL in hyperlipidemic mice. J Lipid Res 48(4):763–767PubMedCrossRefGoogle Scholar
  20. 20.
    Hung G et al (2013) Characterization of target mRNA reduction through in situ RNA hybridization in multiple organ systems following systemic antisense treatment in animals. Nucleic Acid Therapeut 23(6):369–378CrossRefGoogle Scholar
  21. 21.
    Butler M et al (2005) Spinal distribution and metabolism of 2′-O-(2-methoxyethyl)-modified oligonucleotides after intrathecal administration in rats. Neuroscience 131(3):705–715PubMedCrossRefGoogle Scholar
  22. 22.
    Passini MA et al (2011) Antisense oligonucleotides delivered to the mouse CNS ameliorate symptoms of severe spinal muscular atrophy. Sci Transl Med 3:72ra18PubMedCentralPubMedCrossRefGoogle Scholar
  23. 23.
    Grillone LR, Lanz R (2001) Fomivirsen. Drugs Today (Barc) 37(4):245–255Google Scholar
  24. 24.
    Geary RS, Henry SP, Grillone LR (2002) Fomivirsen: clinical pharmacology and potential drug interactions. Clin Pharmacokinet 41(4):255–260PubMedCrossRefGoogle Scholar
  25. 25.
    Seth PP et al (2010) Synthesis and biophysical evaluation of 2′,4′-constrained 2′O-methoxyethyl and 2′,4′-constrained 2′O-ethyl nucleic acid analogues. J Org Chem 75(5):1569–1581PubMedCrossRefGoogle Scholar
  26. 26.
    Ostergaard ME et al (2013) Rational design of antisense oligonucleotides targeting single nucleotide polymorphisms for potent and allele selective suppression of mutant Huntingtin in the CNS. Nucleic Acids Res 41(21):9634–9650PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Xinying Zong
    • 1
  • Lulu Huang
    • 2
  • Vidisha Tripathi
    • 1
  • Raechel Peralta
    • 2
  • Susan M. Freier
    • 2
  • Shuling Guo
    • 2
  • Kannanganattu V. Prasanth
    • 1
  1. 1.Department of Cell and Developmental BiologyUniversity of Illinois at Urbana-ChampaignUrbana-ChampaignUSA
  2. 2.Isis PharmaceuticalsCarlsbadUSA

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