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 ep isomal and chromosomal targets in mammalian cells. We demonstrated that an RNA-DNA oligonucleotide (RDO) induced heritable correction of a point mutat ion in the tyrosinase gene at the level of genomic sequence, protein, and p henotype of albino mouse melanocytes and albino mouse skin. Such RDOs might hold promise as a therapeutic method for the treatment of skin diseases. H owever, the general application of RDO technology has been hampered by the absence of a standardized system to measure the gene conversion in a partic ular cell type in a rapid and reproducible manner. For this purpose, we est ablished 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, coil beta -galactosidase gene. This sensitive and conven ient assay has been utilized to optimize the design of RDOs and to compare frequencies of gene conversion among different cell types. The general appl ication of the RDO for site-specific gene correction or mutation would bene fit from such mechanistic studies.