Targeted single-base correction by RNA-DNA oligonucleotides.

An oligonucleotide composed of a contiguous stretch of RNA and DNA residues has been developed to facilitate correction of single-base mutations of episomal and chromosomal targets in mammalian cells. We demonstrated that an RNA-DNA oligonucleotide (RDO) induced heritable correction of a point mutation in the tyrosinase gene at the level of genomic sequence, protein, and phenotype of albino mouse melanocytes and albino mouse skin. Such RDOs might hold promise as a therapeutic method for the treatment of skin diseases. However, the general application of RDO technology has been hampered by the absence of a standardized system to measure the gene conversion in a particular cell type in a rapid and reproducible manner. For this purpose, we established an in vitro system in which nuclear extracts from mammalian cells showed RDO-mediated gene correction of a shuttle vector containing a point mutation in the E. coli beta-galactosidase gene. This sensitive and convenient assay has been utilized to optimize the design of RDOs and to compare frequencies of gene conversion among different cell types. The general application of the RDO for site-specific gene correction or mutation would benefit from such mechanistic studies.