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DNA Delivery to Mitochondria: Sequence Specificity and Energy Enhancement

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ABSTRACT

Purpose

Mitochondria are competent for DNA uptake in vitro, a mechanism which may support delivery of therapeutic DNA to complement organelle DNA mutations. We document here key aspects of the DNA import process, so as to further lay the ground for mitochondrial transfection in intact cells.

Methods

We developed DNA import assays with isolated mitochondria from different organisms, using DNA substrates of various sequences and sizes. Further import experiments investigated the possible role of ATP and protein phosphorylation in the uptake process. The fate of adenine nucleotides and the formation of phosphorylated proteins were analyzed.

Results

We demonstrate that the efficiency of mitochondrial uptake depends on the sequence of the DNA to be translocated. The process becomes sequence-selective for large DNA substrates. Assays run with a natural mitochondrial plasmid identified sequence elements which promote organellar uptake. ATP enhances DNA import and allows tight integration of the exogenous DNA into mitochondrial nucleoids. ATP hydrolysis has to occur during the DNA uptake process and might trigger phosphorylation of co-factors.

Conclusions

Our data contribute critical information to optimize DNA delivery into mitochondria and open the prospect of targeting whole mitochondrial genomes or complex constructs into mammalian organelles in vitro and in vivo.

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Abbreviations

bp:

base-pair

BSA:

bovine serum albumin

CCCP:

carbonyl cyanide m-chlorophenylhydrazone

DEAE-cellulose:

diethylaminoethyl-cellulose

EDTA:

ethylenediamine tetraacetic acid

EGTA:

ethylene glycol tetraacetic acid

kb:

kilobase-pair

mtDNA:

mitochondrial DNA

OXPHOS:

oxidative phosphorylation

PCR:

polymerase chain reaction

PEI-cellulose:

polyethylenimine-cellulose

PMSF:

phenylmethylsulfonyl fluoride

SDS-PAGE:

sodium dodecyl sulfate-polyacrylamide gel electrophoresis

TLC:

thin-layer chromatography

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ACKNOWLEDGMENTS & DISCLOSURES

We wish to thank Philippe Hammann for mass spectrometry analyses. The mitochondrial transfection project was funded by the French Centre National de la Recherche Scientifique (CNRS, UPR2357) through institutional funding and international cooperation programs, the Université de Strasbourg (UdS), the Agence Nationale de la Recherche (ANR-06-MRAR-037-02), the Welcome Trust, the Russian Academy of Sciences (RAS) and the Russian Foundation for Basic Research (RFBR).

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Authors and Affiliations

  1. Institut de Biologie Moléculaire des Plantes, CNRS and Université de Strasbourg, 12 rue du Général Zimmer, 67084, Strasbourg, France

    Noha Ibrahim, Anne Cosset, Milana Koulintchenko, André Dietrich & Frédérique Weber-Lotfi

  2. Mitochondrial Research Group, Institute for Aging & Health Medical School, Newcastle University, Framlington Place, Newcastle upon Tyne, NE2 4HH, UK

    Noha Ibrahim, Milana Koulintchenko & Robert N. Lightowlers

  3. Plant Genome Research Unit, National Institute of Agrobiological Sciences, 2-1-2 Kan-non-dai, Tsukuba, 305–8602, Japan

    Hirokazu Handa

  4. Siberian Institute of Plant Physiology and Biochemistry, Russian Academy of Sciences, Ul. Lermontova 132, Irkutsk, 664033, Russia

    Milana Koulintchenko & Yuri Konstantinov

Authors
  1. Noha Ibrahim
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  2. Hirokazu Handa
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  3. Anne Cosset
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  4. Milana Koulintchenko
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  5. Yuri Konstantinov
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  6. Robert N. Lightowlers
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  7. André Dietrich
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  8. Frédérique Weber-Lotfi
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Corresponding author

Correspondence to André Dietrich.

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Ibrahim, N., Handa, H., Cosset, A. et al. DNA Delivery to Mitochondria: Sequence Specificity and Energy Enhancement. Pharm Res 28, 2871–2882 (2011). https://doi.org/10.1007/s11095-011-0516-4

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  • DOI: https://doi.org/10.1007/s11095-011-0516-4

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