Pharmaceutical Research

, 28:2871

DNA Delivery to Mitochondria: Sequence Specificity and Energy Enhancement

  • Noha Ibrahim
  • Hirokazu Handa
  • Anne Cosset
  • Milana Koulintchenko
  • Yuri Konstantinov
  • Robert N. Lightowlers
  • André Dietrich
  • Frédérique Weber-Lotfi
Research Paper

DOI: 10.1007/s11095-011-0516-4

Cite this article as:
Ibrahim, N., Handa, H., Cosset, A. et al. Pharm Res (2011) 28: 2871. doi:10.1007/s11095-011-0516-4

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.

KEY WORDS

DNA importmitochondrial diseasemitochondrial plasmidorganelle transfectionprotein phosphorylation

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

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Noha Ibrahim
    • 1
    • 2
  • Hirokazu Handa
    • 3
  • Anne Cosset
    • 1
  • Milana Koulintchenko
    • 1
    • 2
    • 4
  • Yuri Konstantinov
    • 4
  • Robert N. Lightowlers
    • 2
  • André Dietrich
    • 1
  • Frédérique Weber-Lotfi
    • 1
  1. 1.Institut de Biologie Moléculaire des PlantesCNRS and Université de StrasbourgStrasbourgFrance
  2. 2.Mitochondrial Research Group, Institute for Aging & Health Medical SchoolNewcastle UniversityNewcastle upon TyneUK
  3. 3.Plant Genome Research UnitNational Institute of Agrobiological SciencesTsukubaJapan
  4. 4.Siberian Institute of Plant Physiology and BiochemistryRussian Academy of SciencesIrkutskRussia