DNA pairing is a prerequisite step and initiates a critical intermediate by the oligonucleotide in the process of targeted nucleotide exchange
Targeted gene repair consists of at least two major mechanistic steps, the pairing of an oligonucleotide to a site bearing DNA sequence complementarity followed by a nucleotide exchange reaction directed by the oligonucleotide. Various oligonucleotides have been successful in directing the repair of genetic mutations in yeast, plant, and mammalian cells, and show great potential in gene therapy applications. The success of oligonucleotide-directed gene repair relies on the homologous alignment of oligonucleotides with complementary sequences in the target gene, and is dependent on the formation of a highly stable pairing complex. Gene repair activity of an E. coli strain lacking RECA DNA pairing function was rescued by using purified RecA protein to catalyze the assimilation of various oligonucleotide vectors in multiple plasmid systems including either mutant kanamycin or tetracycline resistance genes in vitro. Structural limitations placed on the template indicate correction is influenced significantly by the positioning and modification of the oligonucleotides; data also suggest primer extension of the oligonucleotide does not play a significant role in the gene repair mechanism. Various heterologous oligonucleotides designed to target a stably integrated (mutant) EGFP gene and used to direct the repair of a single base mutation in mammalian DLD-1 cells produced a plethora of effects that have helped us understand the mechanism of repair. The efficiency of correction is influenced by the degree of DNA sequence homology existing between the oligonucleotide and target gene. The negative impact of heterology is dependent on the type of DNA sequence inserted and on the size of the heterologous region. Wild-type levels of repair are restored only when the heterologous sequence is palindromic and adopts a secondary structure. These results reveal the existence of a directional-specific repair pathway that relies on the pairing stability of a bilateral complex and emphasize the importance of sequence homology between pairing partners for efficient catalysis of gene repair. The use of heterologous oligonucleotides to direct gene repair has led to studies involving the role of the mismatch repair system in gene repair, the issue of oligonucleotide inheritance, as well as the use of oligonucleotides as aptamers in targeting Huntington's disease.