Targeted gene correction by small single-stranded oligonucleotides in mammalian cells.
We demonstrate that relatively short single-stranded oligodeoxynucleotides, 25-61 bases homologous to the target sequence except for a single mismatch to the targeted base, are capable of correcting a single point mutation (G to A) in the mutant beta-galactosidase gene, in nuclear extracts, episome, and chromosome of mammalian cells, with correction rates of approximately 0.05%, 1% and 0.1%, respectively. Surprisingly, these short single-stranded oligonucleotides (ODN) showed a similar gene correction frequency to chimeric RNA-DNA oligonucleotide, measured using the same system. The in vitro gene correction induced by ODN in nuclear extracts was not dependent on the length or polarity of the oligonucleotide. In contrast, the episomal and chromosomal gene corrections were highly dependent on the ODN length and polarity. ODN with a homology of 45 nucleotides showed the highest frequency and ODN with antisense orientation showed a 1000-fold higher frequency than sense orientation, indicating a possible influence of transcription on gene correction. Deoxyoligonucleotides showed a higher frequency of gene correction than ribo-oligonucleotides of the identical sequence. These results show that a relatively short ODN can make a sequence-specific change in the target sequence in mammalian cells, at a similar frequency as the chimeric RNA-DNA oligonucleotide.
Igoucheva
O
O
Department of Dermatology and Cutaneous Biology, Department of Biochemistry and Molecular Pharmacology, Jefferson Institute of Molecular Medicine, Thomas Jefferson University and Jefferson Medical College, Philadelphia, PA 19107, USA.
Alexeev
V
V
Yoon
K
K
D000818:Animals; D020137:Base Pair Mismatch / Q000235:genetics; D001483:Base Sequence; D016466:CHO Cells; D006224:Cricetinae; D004260:DNA Repair / Q000235:genetics*; D004278:DNA, Superhelical / Q000235:genetics; D018390:Gene Targeting / Q000379:methods*; D015316:Genetic Therapy / Q000379:methods; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000235:genetics* / Q000494:pharmacology; D010957:Plasmids / Q000235:genetics; D017354:Point Mutation; D012150:Polymorphism, Restriction Fragment Length; D014162:Transfection; D001616:beta-Galactosidase / Q000235:genetics
D004278:DNA, Superhelical; D009841:Oligonucleotides; D001616:beta-Galactosidase