Targeted Gene Correction: Synthesis and Characterization of Double-Hairpin 2′-O-Methyl RNA/DNA Chimera Oligonucleotides

Abstract

A series of 2′-O-methyl RNA/DNA chimera oligonucleotides were synthesized with a double-hairpin structural motif. Liposome formulated delivery of the chimeras effected targeted, high conversion of mutant alleles in mammalian cell culture. The chimera oligonucleotides were prepared with DNA and 2′-OMe RNA phosphoramidite nucleoside monomers on the ABI 394 synthesizer.

Notes

Oligonucleotides were synthesized on an ABI 394 DNA/RNA Synthesizer with Applied Biosystems reagents. Deprotection was conducted in 4 ml glass vials with Teflon lined caps. Considerable pressure results from heating enclosed concentrated ammonium hydroxide solutions. Hot vials should be handled behind a shield with padded gloves.

The 2′-O-Methyl G^dmf phosphoramidite occasionally loses coupling efficiency after several days on the synthesizer

“Evaluating and Isolating Synthetic Oligonucleotides”, Applied Biosystems, Foster City, California, 1992, available upon request