caa a22 4500 445314419 CHVBK 20180317142641.0 cr unu---uuuuu 170323e20111101xx s 000 0 eng 10.1007/s11095-011-0516-4 doi (NATIONALLICENCE)springer-10.1007/s11095-011-0516-4 DNA Delivery to Mitochondria: Sequence Specificity and Energy Enhancement [Elektronische Daten] [Noha Ibrahim, Hirokazu Handa, Anne Cosset, Milana Koulintchenko, Yuri Konstantinov, Robert Lightowlers, André Dietrich, Frédérique Weber-Lotfi] 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. Springer Science+Business Media, LLC, 2011 DNA import nationallicence mitochondrial disease nationallicence mitochondrial plasmid nationallicence organelle transfection nationallicence protein phosphorylation nationallicence bp : base-pair nationallicence BSA : bovine serum albumin nationallicence CCCP : carbonyl cyanide m-chlorophenylhydrazone nationallicence DEAE-cellulose : diethylaminoethyl-cellulose nationallicence EDTA : ethylenediamine tetraacetic acid nationallicence EGTA : ethylene glycol tetraacetic acid nationallicence kb : kilobase-pair nationallicence mtDNA : mitochondrial DNA nationallicence OXPHOS : oxidative phosphorylation nationallicence PCR : polymerase chain reaction nationallicence PEI-cellulose : polyethylenimine-cellulose nationallicence PMSF : phenylmethylsulfonyl fluoride nationallicence SDS-PAGE : sodium dodecyl sulfate-polyacrylamide gel electrophoresis nationallicence TLC : thin-layer chromatography nationallicence Ibrahim Noha Institut de Biologie Moléculaire des Plantes, CNRS and Université de Strasbourg, 12 rue du Général Zimmer, 67084, Strasbourg, France aut Handa Hirokazu Plant Genome Research Unit, National Institute of Agrobiological Sciences, 2-1-2 Kan-non-dai, 305-8602, Tsukuba, Japan aut Cosset Anne Institut de Biologie Moléculaire des Plantes, CNRS and Université de Strasbourg, 12 rue du Général Zimmer, 67084, Strasbourg, France aut Koulintchenko Milana Institut de Biologie Moléculaire des Plantes, CNRS and Université de Strasbourg, 12 rue du Général Zimmer, 67084, Strasbourg, France aut Konstantinov Yuri Siberian Institute of Plant Physiology and Biochemistry, Russian Academy of Sciences, Ul. Lermontova 132, 664033, Irkutsk, Russia aut Lightowlers Robert Mitochondrial Research Group, Institute for Aging & Health Medical School, Newcastle University, Framlington Place, NE2 4HH, Newcastle upon Tyne, UK aut Dietrich André Institut de Biologie Moléculaire des Plantes, CNRS and Université de Strasbourg, 12 rue du Général Zimmer, 67084, Strasbourg, France aut Weber-Lotfi Frédérique Institut de Biologie Moléculaire des Plantes, CNRS and Université de Strasbourg, 12 rue du Général Zimmer, 67084, Strasbourg, France aut Pharmaceutical Research Springer US; http://www.springer-ny.com 28/11(2011-11-01), 2871-2882 0724-8741 28:11<2871 2011 28 11095 https://doi.org/10.1007/s11095-011-0516-4 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/s11095-011-0516-4 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Ibrahim Noha Institut de Biologie Moléculaire des Plantes, CNRS and Université de Strasbourg, 12 rue du Général Zimmer, 67084, Strasbourg, France aut NATIONALLICENCE 700 1- Handa Hirokazu Plant Genome Research Unit, National Institute of Agrobiological Sciences, 2-1-2 Kan-non-dai, 305-8602, Tsukuba, Japan aut NATIONALLICENCE 700 1- Cosset Anne Institut de Biologie Moléculaire des Plantes, CNRS and Université de Strasbourg, 12 rue du Général Zimmer, 67084, Strasbourg, France aut NATIONALLICENCE 700 1- Koulintchenko Milana Institut de Biologie Moléculaire des Plantes, CNRS and Université de Strasbourg, 12 rue du Général Zimmer, 67084, Strasbourg, France aut NATIONALLICENCE 700 1- Konstantinov Yuri Siberian Institute of Plant Physiology and Biochemistry, Russian Academy of Sciences, Ul. Lermontova 132, 664033, Irkutsk, Russia aut NATIONALLICENCE 700 1- Lightowlers Robert Mitochondrial Research Group, Institute for Aging & Health Medical School, Newcastle University, Framlington Place, NE2 4HH, Newcastle upon Tyne, UK aut NATIONALLICENCE 700 1- Dietrich André Institut de Biologie Moléculaire des Plantes, CNRS and Université de Strasbourg, 12 rue du Général Zimmer, 67084, Strasbourg, France aut NATIONALLICENCE 700 1- Weber-Lotfi Frédérique Institut de Biologie Moléculaire des Plantes, CNRS and Université de Strasbourg, 12 rue du Général Zimmer, 67084, Strasbourg, France aut NATIONALLICENCE 773 0- Pharmaceutical Research Springer US; http://www.springer-ny.com 28/11(2011-11-01), 2871-2882 0724-8741 28:11<2871 2011 28 11095 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer