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Characterization of Peptide–Oligonucleotide Complexes Using Electron Microscopy, Dynamic Light Scattering, and Protease Resistance Assay

  • Kärt Padari
  • Ly Porosk
  • Piret Arukuusk
  • Margus PoogaEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2036)

Abstract

Cationic peptides designed for cellular delivery of nucleic acid molecules form noncovalent nanocomplexes with negatively charged oligonucleotides (ON). The electrostatically associated complexes are further compacted by hydrophobic interactions yielding nanoparticles (NP) of homogeneous shape and size that are efficiently taken up by cells. The shape and size of NP often correlate with the biological activity of delivered ON inside cells; and the stability and accessibility of NP in biological fluids govern its circulation in organism and the cellular uptake. Therefore, here we provide protocols for characterizing the shape and size and surface charge of peptide/ON NP by negative staining transmission electron microscopy (TEM) and dynamic light scattering (DLS) respectively, and analysis of NP stability against proteolytic degradation.

Key words

Nucleic acid delivery Peptide–nucleic acid nanocomplexes CPP–oligonucleotide nanoparticles Negative staining TEM CPP nanoparticle DLS Protease resistance assay Oligonucleotide therapeutics 

Notes

Acknowledgments

This study was supported by the Estonian Ministry of Education and Research grant 0180019s11, PUT1617P (K.P. and M.P.) and IUT20-26 (L.P. and P.A.) from the Estonian Research Council. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Kärt Padari
    • 1
  • Ly Porosk
    • 2
  • Piret Arukuusk
    • 2
  • Margus Pooga
    • 1
    • 2
    Email author
  1. 1.Institute of Molecular and Cell BiologyUniversity of TartuTartuEstonia
  2. 2.Institute of TechnologyUniversity of TartuTartuEstonia

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