Skip to main content
SpringerLink
Log in

Lipid-Modified Peptide Nucleic Acids: Synthesis and Application to Programmable Liposome Fusion

  • Protocol
  • First Online:
Peptide Nucleic Acids

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

Abstract

Peptide nucleic acids (PNAs) can be modified with aliphatic lipid chains and designed to be water soluble and able to spontaneously insert into phospholipid bilayers. Liposomes with 1.5% negatively charged POPG can be driven to fuse and mix their inner content volumes via functionalization with such lipidated peptide nucleic acids (LiPNAs). During fusion, only low amounts of leakage occur (<5%). We describe here the synthesis and purification of such LiPNAs using an automated peptide synthesizer and the preparation of LiPNA functionalized liposomes. Further, we describe the measurement of LiPNA-induced fusion using a fluorescence-based assay for the content mixing between a liposome population with an encapsulated self-quenching fluorescent dye (SRB) and a buffer-filled liposome population.

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

    Article  CAS  Google Scholar 

  2. Nielsen PE, Haaima G (1997) Peptide nucleic acid (PNA). A DNA mimic with a pseudopeptide backbone. Chem Soc Rev 26:73–78. https://doi.org/10.1039/CS9972600073

    Article  CAS  Google Scholar 

  3. Ljungstrøm T, Knudsen H, Nielsen PE (1999) Cellular uptake of adamantyl conjugated peptide nucleic acids. Bioconjug Chem 10:965–972. https://doi.org/10.1021/bc990053+

    Article  CAS  PubMed  Google Scholar 

  4. Vernille JP, Kovell LC, Schneider JW (2004) Peptide nucleic acid (PNA) amphiphiles: synthesis, self-assembly, and duplex stability. Bioconjug Chem 15:1314–1321. https://doi.org/10.1021/bc049831a

    Article  CAS  PubMed  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  6. Nielsen PE (2004) PNA technology. Mol Biotechnol 26:233–248. https://doi.org/10.1385/MB:26:3:233

    Article  CAS  PubMed  Google Scholar 

  7. Marques BF, Schneider JW (2005) Sequence-specific binding of DNA to liposomes containing di-alkyl peptide nucleic acid (PNA) amphiphiles. Langmuir 21:2488–2494. https://doi.org/10.1021/la047962u

    Article  CAS  PubMed  Google Scholar 

  8. Lygina AS, Meyenberg K, Jahn R, Diederichsen U (2011) Transmembrane domain peptide/peptide nucleic acid hybrid as a model of a SNARE protein in vesicle fusion. Angew Chem Int Ed 50:8597–8601

    Article  CAS  Google Scholar 

  9. Vogel S et al (2001) Moenomycin analogues with long-chain amine lipid parts from reductive aminations. Tetrahedron 57:4147–4160. https://doi.org/10.1016/S0040-4020(01)00306-4

    Article  CAS  Google Scholar 

  10. Vogel S, Stembera K, Hennig L, Findeisen M, Giesa S, Welzel P, Lampilas M (2001) Moenomycin analogues with modified lipid side chains from indium-mediated Barbier-type reactions. Tetrahedron 57:4139–4146. https://doi.org/10.1016/S0040-4020(01)00301-5

    Article  CAS  Google Scholar 

  11. Miglietta G, Picco R, Vogel S, Wengel J, Xodo LE (2018) MicroRNA therapeutics: design of single-stranded miR-216b mimics to target KRAS in pancreatic cancer cells. RNA Biol 15:1273–1285. https://doi.org/10.1080/15476286.2018.1526536

    Article  PubMed  PubMed Central  Google Scholar 

  12. Cogoi S, Jakobsen U, Pedersen EB, Vogel S, Xodo LE (2016) Lipid-modified G4-decoy oligonucleotide anchored to nanoparticles: delivery and bioactivity in pancreatic cancer cells. Sci Rep 6:38468. https://doi.org/10.1038/srep38468

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Rohr K, Vogel S (2006) Polyaza crown ethers as non-nucleosidic building blocks in DNA conjugates: synthesis and remarkable stabilization of dsDNA. Chembiochem 7:463–470. https://doi.org/10.1002/cbic.200500392

    Article  CAS  PubMed  Google Scholar 

  14. Jakobsen U, Simonsen AC, Vogel S (2008) DNA-controlled assembly of soft nanoparticles. J Am Chem Soc 130:10462–10463. https://doi.org/10.1021/ja8030054

    Article  CAS  PubMed  Google Scholar 

  15. Rabe A, Löffler PMG, Ries O, Vogel S (2017) Programmable fusion of liposomes mediated by lipidated PNA. Chem Commun 53:11921–11924. https://doi.org/10.1039/C7CC06058K

    Article  CAS  Google Scholar 

  16. Pfeiffer I, Höök F (2004) Bivalent cholesterol-based coupling of oligonucletides to lipid membrane assemblies. J Am Chem Soc 126:10224–10225. https://doi.org/10.1021/ja048514b

    Article  CAS  PubMed  Google Scholar 

  17. Marsden HR, Elbers NA, Bomans PHH, Sommerdijk NAJM, Kros A (2009) A reduced SNARE model for membrane fusion. Angew Chem Int Ed Engl 48:2330–2333. https://doi.org/10.1002/anie.200804493

    Article  CAS  Google Scholar 

  18. Ma M, Bong D (2013) Controlled fusion of synthetic lipid membrane vesicles. Acc Chem Res 46:2988–2997. https://doi.org/10.1021/ar400065m

    Article  CAS  PubMed  Google Scholar 

  19. Marsden HR, Tomatsu I, Kros A (2011) Model systems for membrane fusion. Chem Soc Rev 40:1572–1585. https://doi.org/10.1039/c0cs00115e

    Article  CAS  PubMed  Google Scholar 

  20. Pick H, Alves AC, Vogel H (2018) Single-vesicle assays using liposomes and cell-derived vesicles: from modeling complex membrane processes to synthetic biology and biomedical applications. Chem Rev 118:8598–8654. https://doi.org/10.1021/acs.chemrev.7b00777

    Article  CAS  PubMed  Google Scholar 

  21. Christensen SM, Bolinger P-Y, Hatzakis NS, Mortensen MW, Stamou D (2012) Mixing subattolitre volumes in a quantitative and highly parallel manner with soft matter nanofluidics. Nat Nanotechnol 7:51–55

    Article  CAS  Google Scholar 

  22. Oude Blenke EE, van den Dikkenberg J, van Kolck B, Kros A, Mastrobattista E (2016) Coiled coil interactions for the targeting of liposomes for nucleic acid delivery. Nanoscale 8:8955–8965. https://doi.org/10.1039/c6nr00711b

    Article  CAS  PubMed  Google Scholar 

  23. Yang J, Bahreman A, Daudey G, Bussmann J, Olsthoorn RCL, Kros A (2016) Drug delivery via cell membrane fusion using lipopeptide modified liposomes. ACS Cent Sci 2:621–630. https://doi.org/10.1021/acscentsci.6b00172

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Sun L et al (2018) Guiding protein delivery into live cells using DNA-programmed membrane fusion. Chem Sci 9:5967–5975. https://doi.org/10.1039/C8SC00367J

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Fasshauer D, Otto H, Eliason WK, Jahn R, Brünger AT (1997) Structural changes are associated with soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor complex formation. J Biol Chem 272:28036–28041. https://doi.org/10.1074/jbc.272.44.28036

    Article  CAS  PubMed  Google Scholar 

  26. Söllner T, Bennett MK, Whiteheart SW, Scheller RH, Rothman JE (1993) A protein assembly-disassembly pathway in vitro that may correspond to sequential steps of synaptic vesicle docking, activation, and fusion. Cell 75:409–418

    Article  Google Scholar 

  27. Wehland JD, Lygina AS, Kumar P, Guha S, Hubrich BE, Jahn R, Diederichsen U (2016) Role of the transmembrane domain in SNARE protein mediated membrane fusion: peptide nucleic acid/peptide model systems. Mol Biosyst 12:2770. https://doi.org/10.1039/c6mb00294c

    Article  CAS  PubMed  Google Scholar 

  28. Han X, Wang CT, Bai J, Chapman ER, Jackson MB (2004) Transmembrane segments of syntaxin line the fusion pore of Ca2+-triggered exocytosis. Science 304:289–292

    Article  CAS  Google Scholar 

  29. Chernomordik LV, Kozlov MM (2008) Mechanics of membrane fusion. Nat Struct Mol Biol 15:675

    Article  CAS  Google Scholar 

  30. Martens S, McMahon HT (2008) Mechanisms of membrane fusion: disparate players and common principles. Nat Rev Mol Cell Biol 9:543–556. https://doi.org/10.1038/nrm2417

    Article  CAS  PubMed  Google Scholar 

  31. Kozlov MM, Chernomordik LV (2015) Membrane tension and membrane fusion. Curr Opin Struct Biol 33:61–67. https://doi.org/10.1016/j.sbi.2015.07.010

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Kashiwada A, Tsuboi M, Matsuda K (2011) Target-selective one-way membrane fusion system based on a pH-responsive coiled coil assembly at the Interface of liposomal vesicles. Langmuir 27:1403–1408

    Article  CAS  Google Scholar 

  33. Stengel G, Zahn R, Höök F (2007) DNA-induced programmable fusion of phospholipid vesicles. J Am Chem Soc 129:9584–9585. https://doi.org/10.1021/ja073200k

    Article  CAS  PubMed  Google Scholar 

  34. Meng Z et al (2017) Efficient fusion of liposomes by nucleobase quadruple-anchored DNA. Chem Eur J 23:9391. https://doi.org/10.1002/chem.201701379

    Article  CAS  PubMed  Google Scholar 

  35. Ries O, Löffler PMG, Vogel S (2015) Convenient synthesis and application of versatile nucleic acid lipid membrane anchors in the assembly and fusion of liposomes. Org Biomol Chem 13:9673–9680. https://doi.org/10.1039/c5ob01207d

    Article  CAS  PubMed  Google Scholar 

  36. Meyenberg K, Lygina AS, van den Bogaart G, Jahn R, Diederichsen U (2011) SNARE derived peptide mimic inducing membrane fusion. Chem Commun 47:9405–9407. https://doi.org/10.1039/c1cc12879e

    Article  CAS  Google Scholar 

  37. Evans E (1991) Entropy-driven tension in vesicle membranes and unbinding of adherent vesicles. Langmuir 7:1900–1908

    Article  CAS  Google Scholar 

  38. Kozlovsky Y, Kozlov MM (2002) Stalk model of membrane fusion: solution of energy crisis. Biophys J 82:882–895. https://doi.org/10.1016/S0006-3495(02)75450-7

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Francois-Martin C, Rothman JE, Pincet F (2017) Low energy cost for optimal speed and control of membrane fusion. Proc Natl Acad Sci U S A 114:1238–1241. https://doi.org/10.1073/pnas.1621309114

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Parolini L, Mognetti BM, Kotar J, Eiser E, Cicuta P, Di Michele L (2015) Volume and porosity thermal regulation in lipid mesophases by coupling mobile ligands to soft membranes. Nat Commun 6:5948. https://doi.org/10.1038/ncomms6948

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Vogel S (2015) DNA-controlled assembly of soft nanoparticles. In: Stulz E, Clever H (eds) DNA in supramolecular chemistry and nanotechnology. John Wiley & Sons, Ltd., Chichester, pp 397–409. https://doi.org/10.1002/9781118696880.ch5.4

    Chapter  Google Scholar 

  42. Hadorn M, Boenzli E, Sorensen KT, De Lucrezia D, Hanczyc MM, Yomo T (2013) Defined DNA-mediated assemblies of gene-expressing giant unilamellar vesicles. Langmuir 29:15309–15319. https://doi.org/10.1021/la402621r

    Article  CAS  PubMed  Google Scholar 

  43. Brodersen N et al (2007) Nucleosides with 5′-fixed lipid groups – synthesis and anchoring in lipid membranes. Eur J Org Chem 2007:6060–6069. https://doi.org/10.1002/ejoc.200700521

    Article  CAS  Google Scholar 

  44. Gissot A, Camplo M, Grinstaff MW, Barthelemy P (2008) Nucleoside, nucleotide and oligonucleotide based amphiphiles: a successful marriage of nucleic acids with lipids. Org Biomol Chem 6:1324–1333. https://doi.org/10.1039/B719280K

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Schade M, Berti D, Huster D, Herrmann A, Arbuzova A (2014) Lipophilic nucleic acids — a flexible construction kit for organization and functionalization of surfaces. Adv Colloid Interf Sci 208:235–251. https://doi.org/10.1016/j.cis.2014.02.019

    Article  CAS  Google Scholar 

  46. Löffler PMG, Ries O, Rabe A, Okholm AH, Thomsen RP, Kjems J, Vogel S (2017) A DNA-programmed liposome fusion cascade. Angew Chem Int Ed 56:13228–13231. https://doi.org/10.1002/anie.201703243

    Article  CAS  Google Scholar 

  47. Stengel G, Simonsson L, Campbell RA, Höök F (2008) Determinants for membrane fusion induced by cholesterol-modified DNA zippers. J Phys Chem B 112:8264–8274. https://doi.org/10.1021/jp802005b

    Article  CAS  PubMed  Google Scholar 

  48. Chan YHM, van Lengerich B, Boxer SG (2008) Lipid-anchored DNA mediates vesicle fusion as observed by lipid and content mixing. Biointerphases 3:Fa17–Fa21. https://doi.org/10.1116/1.2889062

    Article  CAS  PubMed  Google Scholar 

  49. Ries O, Löffler PMG, Rabe A, Malavan JJ, Vogel S (2017) Efficient liposome fusion mediated by lipid-nucleic acid conjugates. Org Biomol Chem 15:8936–8945. https://doi.org/10.1039/C7OB01939D

    Article  CAS  PubMed  Google Scholar 

  50. Zheng TT, Voskuhl J, Versluis F, Zope HR, Tomatsu I, Marsden HR, Kros A (2013) Controlling the rate of coiled coil driven membrane fusion. Chem Commun 49:3649–3651

    Article  CAS  Google Scholar 

  51. Daudey GA, Zope HR, Voskuhl J, Kros A, Boyle AL (2017) Membrane-fusogen distance is critical for efficient coiled-coil-peptide-mediated liposome fusion. Langmuir 33:12443–12452. https://doi.org/10.1021/acs.langmuir.7b02931

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Kyoung M et al (2011) In vitro system capable of differentiating fast Ca2+-triggered content mixing from lipid exchange for mechanistic studies of neurotransmitter release. Proc Natl Acad Sci U S A 108:E304–E313. https://doi.org/10.1073/pnas.1107900108

    Article  PubMed  PubMed Central  Google Scholar 

  53. Versluis F, Voskuhl J, van Kolck B, Zope H, Bremmer M, Albregtse T, Kros A (2013) In situ modification of plain liposomes with lipidated coiled coil forming peptides induces membrane fusion. J Am Chem Soc 135:8057–8062. https://doi.org/10.1021/ja4031227

    Article  CAS  PubMed  Google Scholar 

  54. Avitabile C, Moggio L, D’Andrea LD, Pedone C, Romanelli A (2010) Development of an efficient and low-cost protocol for the manual PNA synthesis by Fmoc chemistry. Tetrahedron Lett 51:3716–3718. https://doi.org/10.1016/j.tetlet.2010.05.026

    Article  CAS  Google Scholar 

  55. Nielsen PE (2004) Peptide nucleic acids: protocols and applications, 2nd edn. Horizon Bioscience, Wymondham

    Google Scholar 

  56. Itoh YH, Itoh T, Kaneko H (1986) Modified Bartlett assay for microscale lipid phosphorus analysis. Anal Biochem 154:200–204

    Article  CAS  Google Scholar 

  57. Orsi M, Essex JW (2013) Physical properties of mixed bilayers containing lamellar and nonlamellar lipids: insights from coarse-grain molecular dynamics simulations. Faraday Discuss 161:249–272

    Article  CAS  Google Scholar 

  58. Carlowitz B (1995) Kunststoff-Tabellen, vol 4, 4th edn. Carl Hanser Verlag, MĂĽnchen, p 168. https://doi.org/10.1002/mawe.19860170412

    Book  Google Scholar 

Download references

Acknowledgments

We thank Dr. Oliver Ries for his contributions to method development and verification. The authors gratefully acknowledge funding by the Biomolecular Nanoscale Engineering Center (BioNEC), a Centre of Excellence funded by The VILLUM Foundation, grant no. VKR022710.

Conflicts of interest: The authors have no conflicts of interest to declare.

Author information

Authors and Affiliations

  1. Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Odense, Denmark

    Philipp M. G. Löffler, Alexander Rabe & Stefan Vogel

Authors
  1. Philipp M. G. Löffler
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Alexander Rabe
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Stefan Vogel
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Stefan Vogel .

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

Löffler, P.M.G., Rabe, A., Vogel, S. (2020). Lipid-Modified Peptide Nucleic Acids: Synthesis and Application to Programmable Liposome Fusion. 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_5

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-0243-0_5

  • 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