The DNA strand of chimeric RNA/DNA oligonucleotides can direct gene repair/conversion activity in mammalian and plant cell-free extracts

Chimeric oligonucleotides (chimeras), consisting of RNA and DNA bases folde d by complementarity into a double hairpin conformation, have been shown to alter or repair single bases in plant and animal genomes. An uninterrupted stretch of DNA bases within the chimera is known to be active in the seque nce alteration while RNA residues aid in complex stability. In this study, the two strands were separated in the hope of defining the role each plays in conversion. Using a series of single-stranded oligonucleotides, comprise d of all RNA or DNA residues and various mixtures, several new structures h ave emerged as viable molecules in nucleotide conversion. When extracts fro m mammalian and plant cells and a genetic readout assay in bacteria are use d, single-stranded oligonucleotides, containing a defined number of thioate backbone modifications, were found,to be more active than the original chi mera structure in the process of gene repair. Single-stranded oligonucleoti des containing fully modified backbones were found to have low repair activ ity and in fact induce mutation. Molecules containing various lengths of mo dified RNA bases (2'-O-methyl) were also found to possess low activity. Tak en together, these results confirm the directionality of nucleotide convers ion by the DNA strand of the chimera and further present a novel, modified single stranded DNA molecule that directs conversion in plant and animal ce ll-free extracts.