A tool for functional plant genomics: Chimeric RNA/DNA oligonucleotides cause in vivo gene-specific mutations

Self-complementary chimeric oligonucleotides (COs) composed of DNA and modi fied RNA residues were evaluated as a means to (i) create stable, site-spec ific base substitutions in a nuclear gene and (ii) introduce a frameshift i n a nuclear transgene in plant cells. To demonstrate the creation of allele -specific mutations in a member of a gene family, COs were designed to targ et the codon for Pro-196 of SuRA, a tobacco acetolactate synthase (ALS) gen e.,Sn amino acid substitution at Pro-196 of ALS confers a herbicide-resista nce phenotype that can be used as a selectable marker in plant cells. COs w ere designed to contain a 25-nt homology domain comprised of a five-deoxyri bonucleotide region (harboring a single base mismatch to the native ALS seq uence) flanked by regions each composed of 10 ribonucleotides, After recove ry of herbicide-resistant tobacco cells on selective medium, DNA sequence a nalyses identified base conversions in the ALS gene at the codon for Pro-19 6, To demonstrate a site-specific insertion of a single base into a targete d gene, COs were used to restore expression of an inactive green fluorescen t protein transgene that had been designed to contain a single base deletio n. Recovery of fluorescent cells confirmed the deletion correction. Our res ults demonstrate the application of a technology to modify individual genet ic loci by catalyzing either a base substitution or a base addition to spec ific nuclear genes; this approach should have great utility in the area of plant functional genomics.