Skip to main content
SpringerLink
Log in

In Vitro Cellular Delivery of Peptide Nucleic Acid (PNA)

  • Protocol
  • First Online:
Peptide Nucleic Acids

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2105))

Abstract

Cellular delivery methods are a prerequisite for cellular studies with PNA. This chapter describes PNA cellular delivery using cell-penetrating peptide (CPP)-PNA conjugates and transfection of PNA-ligand conjugates mediated by cationic lipids. Furthermore, two endosomolytic procedures employing chloroquine treatment or photochemical internalization (PCI) for significantly improving PNA delivery efficacy are described.

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

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Nielsen PE, Egholm M, Berg RH, Buchardt O (1991) Sequence-selective recognition of DNA by strand displacement with a thymine-substituted polyamide. Science 254:1497–1500

    Article  CAS  Google Scholar 

  2. Torchilin VP (2006) Recent approaches to intracellular delivery of drugs and DNA and organelle targeting. Annu Rev Biomed Eng 8:343–375

    Article  CAS  Google Scholar 

  3. Juliano RL (2016) The delivery of therapeutic oligonucleotides. Nucleic Acids Res 44:6518–6548

    Article  Google Scholar 

  4. Doyle DF, Braasch DA, Simmons CG, Janowski BA, Corey DR (2001) Inhibition of gene expression inside cells by peptide nucleic acids: effect of mRNA target sequence, mismatched bases, and PNA length. Biochemistry 40:53–64

    Article  CAS  Google Scholar 

  5. Shiraishi T, Nielsen PE (2012) Nanomolar cellular antisense activity of peptide nucleic acid (PNA) cholic acid (“umbrella”) and cholesterol conjugates delivered by cationic lipids. Bioconjug Chem 23:196–202

    Article  CAS  Google Scholar 

  6. Mae M, Langel U (2006) Cell-penetrating peptides as vectors for peptide, protein and oligonucleotide delivery. Curr Opin Pharmacol 6:509–514

    Article  Google Scholar 

  7. Shiraishi T, Bendifallah N, Nielsen PE (2006) Cellular delivery of polyheteroaromate-peptide nucleic acid conjugates mediated by cationic lipids. Bioconjug Chem 17:189–194

    Article  CAS  Google Scholar 

  8. Shiraishi T, Hamzavi R, Nielsen PE (2008) Subnanomolar antisense activity of phosphonate-peptide nucleic acid (PNA) conjugates delivered by cationic lipids to HeLa cells. Nucleic Acids Res 36:4424–4432

    Article  CAS  Google Scholar 

  9. Ljungstrom T, Knudsen H, Nielsen PE (1999) Cellular uptake of adamantyl conjugated peptide nucleic acids. Bioconjug Chem 10:965–972

    Article  CAS  Google Scholar 

  10. Margus H, Padari K, Pooga M (2012) Cell-penetrating peptides as versatile vehicles for oligonucleotide delivery. Mol Ther 20:525–533

    Article  CAS  Google Scholar 

  11. Jarver P, Langel K, El-Andaloussi S, Langel U (2007) Applications of cell-penetrating peptides in regulation of gene expression. Biochem Soc Trans 35:770–774

    Article  CAS  Google Scholar 

  12. Wang Q, Yin H, Camelliti P, Betts C, Moulton H, Lee H, Saleh AF, Gait MJ, Wood MJ (2010) In vitro evaluation of novel antisense oligonucleotides is predictive of in vivo exon skipping activity for Duchenne muscular dystrophy. J Gene Med 12:354–364

    Article  Google Scholar 

  13. Yin H, Betts C, Saleh AF, Ivanova GD, Lee H, Seow Y, Kim D, Gait MJ, Wood MJ (2010) Optimization of peptide nucleic acid antisense oligonucleotides for local and systemic dystrophin splice correction in the mdx mouse. Mol Ther 18:819–827

    Article  CAS  Google Scholar 

  14. Yin H, Moulton HM, Betts C, Merritt T, Seow Y, Ashraf S, Wang Q, Boutilier J, Wood MJ (2010) Functional rescue of dystrophin-deficient mdx mice by a chimeric peptide-PMO. Mol Ther 18:1822–1829

    Article  CAS  Google Scholar 

  15. McClorey G, Banerjee S (2018) Cell-penetrating peptides to enhance delivery of oligonucleotide-based therapeutics. Biomedicines 6. https://doi.org/10.3390/biomedicines6020051

    Article  Google Scholar 

  16. Lundin P, Johansson H, Guterstam P, Holm T, Hansen M, Langel U, EL Andaloussi S (2008) Distinct uptake routes of cell-penetrating peptide conjugates. Bioconjug Chem 19:2535–2542

    Article  CAS  Google Scholar 

  17. El-Andaloussi S, Johansson HJ, Lundberg P, Langel U (2006) Induction of splice correction by cell-penetrating peptide nucleic acids. J Gene Med 8:1262–1273

    Article  CAS  Google Scholar 

  18. El-Sayed A, Masuda T, Khalil I, Akita H, Harashima H (2009) Enhanced gene expression by a novel stearylated INF7 peptide derivative through fusion independent endosomal escape. J Control Release 138:160–167

    Article  CAS  Google Scholar 

  19. Koppelhus U, Shiraishi T, Zachar V, Pankratova S, Nielsen PE (2008) Improved cellular activity of antisense peptide nucleic acids by conjugation to a cationic peptide-lipid (CatLip) domain. Bioconjug Chem 19:1526–1534

    Article  CAS  Google Scholar 

  20. Hu J, Corey DR (2007) Inhibiting gene expression with peptide nucleic acid (PNA)—peptide conjugates that target chromosomal DNA. Biochemistry 46:7581–7589

    Article  CAS  Google Scholar 

  21. Abes S, Williams D, Prevot P, Thierry A, Gait MJ, Lebleu B (2006) Endosome trapping limits the efficiency of splicing correction by PNA-oligolysine conjugates. J Control Release 110:595–604

    Article  CAS  Google Scholar 

  22. Shiraishi T, Pankratova S, Nielsen PE (2005) Calcium ions effectively enhance the effect of antisense peptide nucleic acids conjugated to cationic tat and oligoarginine peptides. Chem Biol 12:923–929

    Article  CAS  Google Scholar 

  23. Turner JJ, Ivanova GD, Verbeure B, Williams D, Arzumanov AA, Abes S, Lebleu B, Gait MJ (2005) Cell-penetrating peptide conjugates of peptide nucleic acids (PNA) as inhibitors of HIV-1 Tat-dependent trans-activation in cells. Nucleic Acids Res 33:6837–6849

    Article  CAS  Google Scholar 

  24. Shiraishi T, Nielsen PE (2006) Enhanced delivery of cell-penetrating peptide-peptide nucleic acid conjugates by endosomal disruption. Nat Protoc 1:633–636

    Article  CAS  Google Scholar 

  25. Singh T, Murthy ASN, Yang HJ, Im J (2018) Versatility of cell-penetrating peptides for intracellular delivery of siRNA. Drug Deliv 25:1996–2006

    Article  Google Scholar 

  26. Kosuge M, Takeuchi T, Nakase I, Jones AT, Futaki S (2008) Cellular internalization and distribution of arginine-rich peptides as a function of extracellular peptide concentration, serum, and plasma membrane associated proteoglycans. Bioconjug Chem 19:656–664

    Article  CAS  Google Scholar 

  27. Lv H, Zhang S, Wang B, Cui S, Yan J (2006) Toxicity of cationic lipids and cationic polymers in gene delivery. J Control Release 114:100–109

    Article  CAS  Google Scholar 

  28. Mologni L, Marchesi E, Nielsen PE, Gambacorti-Passerini C (2001) Inhibition of promyelocytic leukemia (PML)/retinoic acid receptor-alpha and PML expression in acute promyelocytic leukemia cells by anti-PML peptide nucleic acid. Cancer Res 61:5468–5473

    CAS  Google Scholar 

  29. Fotin-Mleczek M, Fischer R, Brock R (2005) Endocytosis and cationic cell-penetrating peptides—a merger of concepts and methods. Curr Pharm Des 11:3613–3628

    Article  CAS  Google Scholar 

  30. Nakase I, Niwa M, Takeuchi T, Sonomura K, Kawabata N, Koike Y, Takehashi M, Tanaka S, Ueda K, Simpson JC et al (2004) Cellular uptake of arginine-rich peptides: roles for macropinocytosis and actin rearrangement. Mol Ther 10:1011–1022

    Article  CAS  Google Scholar 

  31. Yarani R, Shiraishi T, Nielsen PE (2018) Effective photo-enhancement of cellular activity of fluorophore-octaarginine antisense PNA conjugates correlates with singlet oxygen formation, endosomal escape and chromophore lipophilicity. Sci Rep 8:638

    Article  Google Scholar 

  32. Erbacher P, Roche AC, Monsigny M, Midoux P (1996) Putative role of chloroquine in gene transfer into a human hepatoma cell line by DNA/lactosylated polylysine complexes. Exp Cell Res 225:186–194

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by the Lundbeck Foundation and the Danish Council for Independent Research/Medical Sciences. This chapter is an updated version of a chapter from the previous edition.

Author information

Authors and Affiliations

  1. Department of Cellular and Molecular Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark

    Takehiko Shiraishi, Mahdi Ghavami & Peter E. Nielsen

Authors
  1. Takehiko Shiraishi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Mahdi Ghavami
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Peter E. Nielsen
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. Department of Cellular and Molecular Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark

    Peter E. Nielsen

Rights and permissions

Reprints and permissions

Copyright information

© 2020 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Shiraishi, T., Ghavami, M., Nielsen, P.E. (2020). In Vitro Cellular Delivery of Peptide Nucleic Acid (PNA). In: Nielsen, P. (eds) Peptide Nucleic Acids. Methods in Molecular Biology, vol 2105. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0243-0_10

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-0243-0_10

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0242-3

  • Online ISBN: 978-1-0716-0243-0

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation