A plausible mechanism for gene correction by chimeric oligonucleotides.
Self-complementary chimeric oligonucleotides that consist of DNA and 2'-O-methyl RNA nucleotides arranged in a double-hairpin configuration can elicit a point mutation when targeted to a gene sequence. We have used a series of structurally diverse chimeric oligonucleotides to correct a mutant neomycin phosphotransferase gene in a human cell-free extract. Analysis of structure-activity relationships demonstrates that the DNA strand of the chimeric oligonucleotide acts as a template for high-fidelity gene correction when one of its bases is mismatched to the targeted gene. By contrast, the chimeric strand of the oligonucleotide does not function as a template for gene repair. Instead, it appears to augment the frequency of gene correction by facilitating complex formation with the target. In the presence of RecA protein, each strand of a chimeric oligonucleotide can hybridize with double-stranded DNA to form a complement-stabilized D-loop. This reaction, which may take place by reciprocal four-strand exchange, is not observed with oligonucleotides that lack 2'-O-methyl RNA segments. Preliminary sequencing data suggest that complement-stabilized D-loops may be weakly mutagenic. If so, a low level of random mutagenesis in the vicinity of the chimera binding site may accompany gene repair.
Gamper
H B
HB
Department of Biological Sciences, University of Delaware, Newark, Delaware 19716, USA.
Cole-Strauss
A
A
Metz
R
R
Parekh
H
H
Kumar
R
R
Kmiec
E B
EB
D002474:Cell-Free System; D004247:DNA / Q000138:chemical synthesis / Q000235:genetics; D004260:DNA Repair; D005813:Genes, Synthetic; D006801:Humans; D019868:Kanamycin Kinase / Q000737:chemistry / Q000235:genetics; D009692:Nucleic Acid Heteroduplexes / Q000138:chemical synthesis / Q000235:genetics; D009841:Oligonucleotides / Q000138:chemical synthesis / Q000737:chemistry* / Q000235:genetics*; D017354:Point Mutation; D012313:RNA / Q000138:chemical synthesis / Q000235:genetics; D011938:Rec A Recombinases / Q000737:chemistry
D009692:Nucleic Acid Heteroduplexes; D009841:Oligonucleotides; D012313:RNA; D004247:DNA; D019868:Kanamycin Kinase; D011938:Rec A Recombinases