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Molecular Methods for Validation of the Biological Activity of Peptide Nucleic Acids Targeting MicroRNAs

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miRNA Maturation

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1095))

Abstract

The involvement of microRNAs in human pathologies is a firmly established fact. Accordingly, the pharmacological modulation of their activity appears to be a very appealing issue in the development of new types of drugs (miRNA therapeutics). One of the most interesting issues is the possible development of miRNA therapeutics for development of anti-cancer molecules. In this respect appealing molecules are based on peptide nucleic acids (PNAs), displaying a pseudo-peptide backbone composed of N-(2-aminoethyl)glycine units and found to be excellent candidates for antisense and antigene therapies. The major limit in the use of PNAs for alteration of gene expression is the low uptake by eukaryotic cells. The aim of this chapter is to describe methods for determining the activity of PNAs designed to target oncomiRNAs, using as model system miR-221 and its target p27Kip1 mRNA. The effects of PNAs targeting miR-221 are here presented discussing data obtained using as model system the human breast cancer cell line MDA-MB-231, in which miR-221 is up-regulated and p27Kip1 down-regulated.

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Abbreviations

AEEA:

2-(2-aminoethoxy)ethoxyacetyl spacer

FACS:

Fluorescence-activated cell sorter

FBS:

Fetal bovine serum

Fl:

Fluorescein

PBS:

Phosphate-buffered saline

PNA:

Peptide nucleic acid

3′UTR:

3′-untranslated region

RT-qPCR:

Retro transcription-quantitative polymerase chain reaction

EDTA:

Ethylenediaminetetraacetic acid

SDS:

Sodium dodecyl sulfate

DTT:

Dithiotreithol

TBS:

Tris-buffered saline

HRP:

Horseradish peroxidase

RISC:

RNA-induced silencing complex

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Acknowledgments

This work was partially supported by a grant from MIUR (PRIN09 grant n. 20093N774P “Molecular recognition of microRNA (miR) by modified PNA: from structure to activity”). R.G. is granted by Fondazione Cariparo (Cassa di Risparmio di Padova e Rovigo), by UE ITHANET Project (Infrastructure for the Thalassaemia Research Network), by Telethon (contract GGP10214). This research is also supported by CIB (Interuniversity Consortium for Biotechnologies) and by Associazione Veneta per la Lotta alla Talassemia (AVLT), Rovigo.

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Authors and Affiliations

  1. Department of Life Sciences and Biotechnology, Ferrara University, Ferrara, Italy

    Eleonora Brognara, Enrica Fabbri, Nicoletta Bianchi & Roberto Gambari

  2. Laboratory for the Development of Pharmacological and Pharmacogenomic Therapy of Thalassaemia, Biotechnology Center, Ferrara University, Ferrara, Italy

    Nicoletta Bianchi, Alessia Finotti & Roberto Gambari

  3. Department of Organic Chemistry, Parma University, Parma, Italy

    Roberto Corradini

Authors
  1. Eleonora Brognara
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  2. Enrica Fabbri
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  3. Nicoletta Bianchi
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  4. Alessia Finotti
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  5. Roberto Corradini
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  6. Roberto Gambari
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Editor information

Editors and Affiliations

  1. Institute for Chemistry, Humboldt Universität zu Berlin, Berlin, Berlin, Germany

    Christoph Arenz

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Brognara, E., Fabbri, E., Bianchi, N., Finotti, A., Corradini, R., Gambari, R. (2014). Molecular Methods for Validation of the Biological Activity of Peptide Nucleic Acids Targeting MicroRNAs. In: Arenz, C. (eds) miRNA Maturation. Methods in Molecular Biology, vol 1095. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-703-7_14

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  • DOI: https://doi.org/10.1007/978-1-62703-703-7_14

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-702-0

  • Online ISBN: 978-1-62703-703-7

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