Epoch Pharmaceuticals, Inc.

Kimeragen, Inc.

Webb, Nigel L.

Howard B Gamper, Jr

Rich B Meyer, Jr

Eric B Kmiec

Nigel L Webb

Allyson D Cole-Strauss

Methods and compositions for efficient targeting and modification of target sequences in duplex DNA are provided, utilizing oligonucleotides or oligonucleotide compositions containing two domains. The first domain comprises an entity capable of recognizing a double-stranded DNA sequence. This can be a protein, peptide, antibiotic, minor groove binding agent or a nucleotide sequence capable of triplex formation The second domain, which is covalently joined to the first, is capable of recognizing a single-stranded DNA sequence. This second domain will most often be complementary, in the Watson-Crick sense, to a target sequence in the double-stranded nucleic acid. The second domain can optionally carry one or more modifying groups, capable of causing a mutation, a pre-mutagenic lesion, or some other type of heritable change in the target sequence.

Methods and compositions to facilitate D-loop formation by oligonucleotides

The invention is based on the reaction of Duplex Mutational Vector in a cell-free system containing a cytoplasmic cell extract and a test plasmid. The reaction specifically converts a mutant kan.sup.r gene to recover the resistant phenotype in transformed MutS, RecA deficient bacteria. Using this system a type of Duplex Mutational Vector termed a Non-Chimeric Mutational Vector, having no RNA:DNA hybrid-duplex is shown to be an effective substrate for eukaryotic enzymes. The invention concerns the use of Non-Chimeric Mutational Vectors protected from 3' exonuclease attack in eukaryotic cells. Such protection can be conferred by replacement of a tetrathymidine linker by a nuclease resistant oligonucleotide, such as tetra-2'-O-methyl-uridine, to link the two strands of the recombinagenic oligonucleobase.

Eukaryotic use of non-chimeric mutational vectors

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Philadelphia, PA, United States of America

Howard B Gamper, Jr