A High-Throughput Screening Assay for the Functional Delivery of Splice-Switching Oligonucleotides in Human Melanoma Cells

  • John M. Dean
  • Robert K. DeLong
Part of the Methods in Molecular Biology book series (MIMB, volume 1297)


Since the conception of RNA nanotechnology (Cell, 94:147, 1998), there has been tremendous interest in its application for the functional delivery of RNA into cells. Splice-switching oligonucleotides (SSOs) are an emerging antisense drug class with the ability to therapeutically modify gene expression. A wide variety of chemical modifications have been devised to try to increase the activity and stability of SSOs. Also, as with most nucleic acid therapeutics, delivery into the cell is the major hurdle for in vivo and clinical applications. As a result, various RNA nanoparticles are being constructed for targeted delivery of therapeutics. However, it is difficult to find a practical assay to measure splice-switching activity. Here, we describe a model delivery system that can be used as a convenient, high-throughput assay to quantitatively measure the functional delivery and splicing redirection in a live human melanoma cell line.

Key words

Transfection Alternative splicing Splice-switching oligonucleotides (SSOs) Delivery Nanomaterials Luciferase 


  1. 1.
    Wang ET, Sandberg R, Luo S et al (2008) Alternative isoform regulation in human tissue transcriptomes. Nature 456:470–476CrossRefGoogle Scholar
  2. 2.
    Lopez-Bigas N, Audit B, Ouzounis C et al (2005) Are splicing mutations the most frequent cause of hereditary disease? FEBS Lett 579:1900–1903CrossRefGoogle Scholar
  3. 3.
    Bauman J, Li S, Yang A et al (2010) Anti-tumor activity of splice-switching oligonucleotides. Nucleic Acids Res 38(22):8348–8356CrossRefGoogle Scholar
  4. 4.
    van Deutekom J, Janson A, Ginjaar I et al (2007) Local dystrophin restoration with antisense oligonucleotide. N Engl J Med 357:2677–2686CrossRefGoogle Scholar
  5. 5.
    Mann C, Homeyman K, Cheng A et al (2000) Antisense-induced exon skipping and synthesis of dystrophin in the mdx mouse. Proc Natl Acad Sci U S A 98(1):42–47CrossRefGoogle Scholar
  6. 6.
    Chan J, Lim S, Wong W (2006) Antisense oligonucleotides: from design to therapeutic application. Clin Exp Pharmacol Physiol 33:533–540CrossRefGoogle Scholar
  7. 7.
    Bauman J, Jearawiriyapaisarn N, Kole R (2009) Therapeutic potential of splice-switching oligonucleotides. Oligonucleotides 19(1):1–13CrossRefGoogle Scholar
  8. 8.
    Kurreck J (2003) Antisense technologies: improvement through novel chemical modifications. Eur J Biochem 270:1628–1644CrossRefGoogle Scholar
  9. 9.
    Guo P, Zhang C, Chen C et al (1998) Inter-RNA interaction of phage φ29 pRNA to form a hexameric complex for viral DNA transportation. Mol Cell 2:149–155CrossRefGoogle Scholar
  10. 10.
    Shu Y, Pi F, Sharma A et al (2014) Stable RNA nanoparticles as potential new generation drugs for cancer therapy. Adv Drug Deliv Rev 66:74–89CrossRefGoogle Scholar
  11. 11.
    Kang S, Cho M, Kole R (1998) Up-regulation of luciferase gene expression with antisense oligonucleotides: implications and applications in functional assay development. Biochemistry 37(18):6235–6239CrossRefGoogle Scholar
  12. 12.
    Mäe M, Samir E, Lundin P et al (2009) A stearylated CPP for delivery of splice correcting oligonucleotides using a non-covalent co-incubation strategy. J Control Release 134:221–227CrossRefGoogle Scholar
  13. 13.
    Kotula J, Pratico E, Ming X et al (2012) Aptamer-mediated delivery of splice-switching oligonucleotides to the nuclei of cancer cells. Nucleic Acid Ther 22:187–195Google Scholar
  14. 14.
    Abes S, Williams D, Prevot P et al (2006) Endosome trapping limits the efficiency of splicing correction by PNA-oligolysine conjugates. J Control Release 110:595–604CrossRefGoogle Scholar
  15. 15.
    El-Andaloussi S, Johansson H, Lundberg P et al (2006) Induction of splice correction by cell-penetrating peptide nucleic acids. J Gene Med 8:1262–1273CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Department of Biology and Biomedical SciencesWashington University in St. LouisSt. LouisUSA
  2. 2.Nanotechnology Innovation Center Kansas State, Department of Anatomy and PhysiologyCollege of Veterinary Medicine, Kansas State UniversityManhattanUSA

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