Methods of attaching unprotected oligonucleotides to DNA-binding, fluorescent, or reactive ligands for synthesis of antisense or gene-directed agentsand probes

The article describes the optimized methods for covalent attachment of unpr otected oligonucleotides to functionally important ligands through their te rminal phosphate or thiophosphate, including covalent attachment of one oli gonucleotide to another. A comparative description of methods is presented for selective introduction of the phosphate, thiophosphate, amino, sulfhydr yl, aldehyde, carboxylic, and other groups into the terminal nucleotide usi ng chemical and enzymatic reactions both in aqueous and organic media. Depe nding on their chemical nature, these groups can then interact with electro philic or nucleophilic ligands carrying aliphatic or aromatic amino groups, hydrazido, sulfhydryl, disulfide, carboxylic, hydroxyl, aldehyde, bromo- o r iodoalkyl, isothiocyanate, and other functions. The available methods all ow one to vary the size of the linker between the oligonucleotide and ligan d, its hydrophobicity and stability in acidic or alkaline media. The use of the disulfide bond permits cleavage of the oligonucleotide-ligand linkage in mild conditions.