title: Characterization of chemically modified oligonucleotides targeting a pathogenic mutation in human mitochondrial DNA.
abstract: Defects in mitochondrial genome can cause a wide range of clinical disorders, mainly neuromuscular diseases. Most of the deleterious mitochondrial mutations are heteroplasmic, meaning that wild type and mutated forms of mtDNA coexist in the same cell. Therefore, a shift in the proportion between mutant and wild type molecules could restore mitochondrial functions. The anti-replicative strategy aims to induce such a shift in heteroplasmy by mitochondrial targeting specifically designed molecules in order to inhibit replication of mutant mtDNA. Recently, we developed mitochondrial RNA vectors that can be used to address anti-replicative oligoribonucleotides into human mitochondria and impact heteroplasmy level, however, the effect was mainly transient, probably due to a rapid degradation of RNA molecules. In the present study, we introduced various chemically modified oligonucleotides in anti-replicative RNAs. We show that the most important increase of anti-replicative molecules' lifetime can be achieved by using synthetic RNA-DNA chimerical molecules or by ribose 2'-O-methylation in nuclease-sensitive sites. The presence of inverted thymidine at 3' terminus and modifications of 2'-OH ribose group did not prevent the mitochondrial uptake of the recombinant molecules. All the modified oligonucleotides were able to anneal specifically with the mutant mtDNA fragment, but not with the wild-type one. Nevertheless, the modified oligonucleotides did not cause a significant effect on the heteroplasmy level in transfected transmitochondrial cybrid cells bearing a pathogenic mtDNA deletion, proving to be less efficient than non-modified RNA molecules.
authors.affiliation: Department of Molecular and Cellular Genetics, UMR 7156 Génétique Moléculaire, Génomique, Microbiologie (GMGM), Strasbourg University - CNRS, Strasbourg 67084, France.
authors.forename: Yann
authors.initials: Y
authors.lastname: Tonin
authors.affiliation: Department of Molecular and Cellular Genetics, UMR 7156 Génétique Moléculaire, Génomique, Microbiologie (GMGM), Strasbourg University - CNRS, Strasbourg 67084, France.
authors.forename: Anne-Marie
authors.initials: AM
authors.lastname: Heckel
authors.affiliation: Department of Molecular and Cellular Genetics, UMR 7156 Génétique Moléculaire, Génomique, Microbiologie (GMGM), Strasbourg University - CNRS, Strasbourg 67084, France; Laboratory of RNA Chemistry, Institute of Chemical Biology and Fundamental Medicine SB RAS, Novosibirsk, Russia.
authors.forename: Ilya
authors.initials: I
authors.lastname: Dovydenko
authors.affiliation: Laboratory of RNA Chemistry, Institute of Chemical Biology and Fundamental Medicine SB RAS, Novosibirsk, Russia.
authors.forename: Mariya
authors.initials: M
authors.lastname: Meschaninova
authors.affiliation: Department of Molecular and Cellular Genetics, UMR 7156 Génétique Moléculaire, Génomique, Microbiologie (GMGM), Strasbourg University - CNRS, Strasbourg 67084, France.
authors.forename: Caroline
authors.initials: C
authors.lastname: Comte
authors.affiliation: Laboratory of RNA Chemistry, Institute of Chemical Biology and Fundamental Medicine SB RAS, Novosibirsk, Russia.
authors.forename: Alya
authors.initials: A
authors.lastname: Venyaminova
authors.affiliation: Laboratory of RNA Chemistry, Institute of Chemical Biology and Fundamental Medicine SB RAS, Novosibirsk, Russia.
authors.forename: Dmitrii
authors.initials: D
authors.lastname: Pyshnyi
authors.affiliation: Department of Molecular and Cellular Genetics, UMR 7156 Génétique Moléculaire, Génomique, Microbiologie (GMGM), Strasbourg University - CNRS, Strasbourg 67084, France.
authors.forename: Ivan
authors.initials: I
authors.lastname: Tarassov
authors.affiliation: Department of Molecular and Cellular Genetics, UMR 7156 Génétique Moléculaire, Génomique, Microbiologie (GMGM), Strasbourg University - CNRS, Strasbourg 67084, France. Electronic address: n.entelis@unistra.fr.
authors.forename: Nina
authors.initials: N
authors.lastname: Entelis
mesh_terms: D002478:Cells, Cultured; D002678:Chimera / Q000235:genetics* / Q000378:metabolism; D004272:DNA, Mitochondrial / Q000037:antagonists & inhibitors* / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation; D018740:Genetic Heterogeneity; D005822:Genetic Vectors; D005838:Genotype; D006801:Humans; D040582:Inheritance Patterns; D008928:Mitochondria / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D028361:Mitochondrial Diseases / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D008938:Mitosis; D058990:Molecular Targeted Therapy; D009154:Mutation; D009843:Oligoribonucleotides / Q000138:chemical synthesis / Q000235:genetics* / Q000378:metabolism; D010641:Phenotype
chemical_list: D004272:DNA, Mitochondrial; D009843:Oligoribonucleotides
keywords: Cybrid cells; Mitochondria; Modified oligonucleotides; RNA import; mtDNA heteroplasmy