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NickFects, Phosphorylated Derivatives of Transportan 10 for Cellular Delivery of Oligonucleotides

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Abstract

Oligonucleotide-based gene regulation has a high potential in gene therapy, but the plasma membrane is impermeable for nucleic acid polymers and, consequently, an efficient and non-toxic transfection agent is needed for their delivery into the cell. In this study we present a novel series, NickFects, of chemically modified TP10 peptide-based delivery vectors used for the cellular delivery of single-stranded oligonucleotides. These carriers, obtained by replacement of Ile8 by threonine in stearyl-TP10 and by modifying of tyrosine and/or threonine, respectively, by phosphorylation formed 300–500 nm in size peptide:oligonucleotide nanocomplexes with negative surface charges. The highest splice-correcting effect was obtained when phosphorotiate 2′-O-methyl oligonucleotides were transduced into cells by NickFect1 (NF1) or NickFect2 (NF2). In addition, we also show how a small modification (one or two negative charges) in peptide sequence can affect its ability to deliver ONs into cells and increase their potency in the splicing redirection assay. Our studies demonstrate that NF1 and NF2 have higher transfection efficacy for oligonucleotides as compared to the most commonly used transfection agent Lipofectamine™ 2000 and lead to higher biological response in cells.

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Abbreviations

2′-OMe ON:

Phosphorothioate 2′-O-methyl RNA

SCO:

Splice-correcting oligonucleotides

CPPs:

Cell-penetrating peptides

PBS:

Phosphate buffered saline

TFA:

Trifluoroacetic acid

TP10:

Transportan 10

ON:

Oligonucleotide

SFM:

Serum-free medium

FM:

Serum-containing medium

OD:

Optical density

HOBt:

Hydroxybenzotriazole

HBTU:

O-Benzotriazole-N,N,N′,N′-tetramethyl-uronium-hexafluoro-phosphate

DIEA:

Diisopropylethylamine

TIS:

Triisopropylsilane

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Acknowledgments

The study presented in this article was supported by Swedish Research Council (VR-NT); by the Center for Biomembrane Research, Stockholm; by Cepep Eesti OÜ; by the Knut and Alice Wallenberg’s Foundation; by the EU through the European Regional Development Fund through the Center of Excellence in Chemical Biology and in Genomics, Estonia; by the targeted financing SF0180027s08 and SF0180019s11 from the Estonian Ministry of Education and Research; by the DoRa Program of The European Social Fund; by Archimedes Foundation and by the Estonian Science Foundation (ESF 8705, Mobilitas—MJD64). The authors would also like to thank J. Pae and C. Juks for assistance in peptide synthesis, S. El-Andaloussi for fruitful discussions and M. Kure for an excellent technical assistance in electron microscopy.

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Authors and Affiliations

  1. Laboratory of Molecular Biotechnology, Institute of Technology, University of Tartu, Nooruse 1, 50411, Tartu, Estonia

    Nikita Oskolkov, Piret Arukuusk, Dana-Maria Copolovici & Ülo Langel

  2. Department of Neurochemistry, The Arrhenius Laboratories for Natural Sciences, Stockholm University, Svante Arrheniusväg 21A, 10691, Stockholm, Sweden

    Staffan Lindberg & Ülo Langel

  3. Department of Developmental Biology, Institute of Molecular and Cell Biology, University of Tartu, 51010, Tartu, Estonia

    Helerin Margus, Kärt Padari & Margus Pooga

Authors
  1. Nikita Oskolkov
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  2. Piret Arukuusk
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  3. Dana-Maria Copolovici
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  4. Staffan Lindberg
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  5. Helerin Margus
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  6. Kärt Padari
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  7. Margus Pooga
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  8. Ülo Langel
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Correspondence to Nikita Oskolkov.

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Oskolkov, N., Arukuusk, P., Copolovici, DM. et al. NickFects, Phosphorylated Derivatives of Transportan 10 for Cellular Delivery of Oligonucleotides. Int J Pept Res Ther 17, 147–157 (2011). https://doi.org/10.1007/s10989-011-9252-1

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  • DOI: https://doi.org/10.1007/s10989-011-9252-1

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