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

Abstract

Chimeric oligonucleotides (chimeras), consisting of RNA and DNA bases folded 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 sequence 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, comprised of all RNA or DNA residues and various mixtures, several new structures have emerged as viable molecules in nucleotide conversion. When extracts from mammalian and plant cells and a genetic readout assay in bacteria are used, single-stranded oligonucleotides, containing a defined number of thioate backbone modifications, were found to be more active than the original chimera structure in the process of gene repair. Single-stranded oligonucleotides containing fully modified backbones were found to have low repair activity and in fact induce mutation. Molecules containing various lengths of modified RNA bases (2'-O-methyl) were also found to possess low activity. Taken together, these results confirm the directionality of nucleotide conversion by the DNA strand of the chimera and further present a novel, modified single-stranded DNA molecule that directs conversion in plant and animal cell-free extracts.

Progress and prospects: oligonucleotide-directed gene modification in mouse embryonic stem cells: a route to therapeutic application

“…Chemical modifications such as phosphorothioate (PTO) linkages, 46 or locked nucleic acids, 61 have been added to protect the ssODNs against nucleolytic degradation. However, besides improving the bioavailability of ssODNs, chemical modifications also have deleterious side effects.…”

Commentary: Genetic modification through oligonucleotide-mediated mutagenesis. A GMO regulatory challenge?

“…Although the usefulness of the technique has been first demonstrated in mammalian cells, preliminary studies at the end of the nineties have shown that oligonucleotidemediated mutagenesis is applicable to plants and can induce target gene mutations (Beetham et al, 1999;Gamper et al, 2000;Hohn and Puchta, 1999;Zhu et al, 1999). Successful in vivo gene modification has been demonstrated notably in maize, rice, tobacco and wheat, e.g.…”

Delivery and mechanistic considerations for the production of knock-in mice by single-stranded oligonucleotide gene targeting

“…14 Protective phosophorothioate linkages aid transfections and increase efficiencies. 15 The pairing of ssODNs to their target is more likely if the target is accessible in a single-stranded state. Both transcription 16,17 and replication 8,16,18 have been linked to higher efficiencies.…”