Sub-cellular temporal and spatial distribution of electrotransferred LNA/DNA oligomer


Research Report

J RNAi Gene Silencing (2013), 9, 479-485

doi: jrgsxx

Published online: 15 March 2013

Full Text: (pdf ~1611kb)

Sub-cellular temporal and spatial distribution of electrotransferred LNA/DNA oligomer

Julie Orio †‡, Elisabeth Bellard †‡, Houda Baaziz †‡, Chantal Pichon §, Peter Mouritzen ¥, Marie-Pierre Rols †‡, Justin Teissié †‡, Muriel Golzio †‡* and Sophie Chabot †‡*

†Centre National de la Recherche Scientifique, Institut de Pharmacologie et de Biologie Structurale, BP64182, 205 route de Narbonne, F-31077Toulouse, France

‡Université de Toulouse, UPS, IPBS, F-31077 Toulouse, France

§Centre de Biophysique Moléculaire, CNRS UPR4301, rue Charles Sadron F-45071 Orléans, Cedex 02, Inserm and Université d ’ Orléans, France

¥Exiqon, Skelstedet 16, 2950 Vedbaek, Denmark

*Correspondence toMuriel Golzio, Email: muriel.golzio@ipbs.fr, or Sophie Chabot, Email: sophie.chabot@ipbs.fr, Tel: +33 561 175813/27; Fax: +33 561 175994

Received: 27 November 2012, Revised: 04 March 2013, Accepted: 15 March 2013

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ABSTRACT

RNA interference (RNAi) is considered as a major scientific breakthrough and the number of in vitroRNAi experiments have increased continuously. However, low biological activity and inefficient targeted delivery in vivo have hindered RNAi-based therapy from reaching its full clinical potential. To overcome these hurdles, progresses have been made to develop new technologies optimizing oligonucleotides chemistry on one hand and achieving its effective delivery on the other hand. In this report, we achieved, by using the electropulsation technique (EP), efficient cellular delivery of chemically-modified oligonucleotide: the locked nucleic acid (LNA) / DNA oligomer. We used single cell level confocal fluorescence microscopy to follow the spatial and temporal distribution of electrotransferred cyanine 5 (Cy5)-labeled LNA/DNA oligomer. We observed that EP allowed LNA/DNA oligomer cellular uptake providing the oligomer a rapid access to the cytoplasm of HeLa cells. Few minutes after electrotransfer, Cy5-LNA/DNA oligomer shuttles from cytoplasm to nucleus whereas in absence of pulses application, Cy5-LNA/DNA oligomer was not detected. We then observed a redistribution of the Cy5 fluorescence that accumulated over time into cytoplasmic organelles. To go further and identify these compartments, we used the HeLa GFP-Rab7 cell line to visualize late endosomes, and lysosomal or mitochondrial specific markers. Our results showed that the EP technique allowed direct entry into the cytoplasm of the Cy5-LNA/DNA oligomer bypassing the endocytosic pathway. On the opposite, in absence of pulses application, Cy5-LNA/DNA oligomer was able to enter cells by itself through the endocytosic pathway. We demonstrated that EP is an efficient technique for LNA-based oligonucleotides delivery offering strong advantages by avoiding the endolysosomal compartmentalization, giving a rapid and free access to the cytoplasm and the nucleus where they can find their targets.

Keywords: Electropulsation, Electroporation, RNAi, Locked nucleic acid, Fluorescence microscopy, cyanine 5 labeling

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