A simple and efficient method for attaching amines to the terminal 5'-phosphate of unprotected oligonucleotides or nucleic acids in aqueous solution is described. The method is applicable to low molecular-weight amines, polypeptides, or proteins. The terminal 5'-phosphate of an oligonucleotide or nucleic acid reacts with a water-soluble carbodiimide in imidazole buffer at pH 6 to give good yields of the 5'-phosphorimidazolide. Exposure of the phosphorimidazolide to amine-containing molecules in aqueous solution results in the production of a wide range of stable phosphoramidates in high yield. The exposure of polynucleotides to carbodiimide does not result in significant breakage of phosphodiester bonds or damage to nucleoside bases. The biological activity of a drug resistant plasmid is not affected. The direct condensation of polynucleotides with amines in 1-methylimidazole buffer is also possible. However, it is not a satisfactory preparative method if the ligand is sensitive to carbodiimide.

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Ethylenediaminetetraacetic acid and diethylenetriaminepentaacetic acid have been attached covalently to the 5' terminus of the deoxynucleotide sequence C-A-C-A-A-T-T-C-C-A-C-A-C-A-A-C (16-mer) via an ethylenediamine linker. In the presence of Fe2+ and dithiothreitol, these reagents bring about the hybridization-dependent cleavage of the sequence T-C-G-T-A-T-G-T-T-G-T-G-T-G-G-A-A-T-T-G-T-G-A-G-C-G-G-A-T-A-A-C-A-A-T-T- T (37-mer), a sequence that contains an internal subsequence complementary to the 16-mer. The principal cleavage sites on the 37-mer are about four residues on each side of the terminal phosphate group of the 16-mer.

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We have developed general methods for joining together, via cleavable disulfide bonds, either two unprotected polynucleotides or a polynucleotide and a peptide or protein. To join two oligonucleotides, each is first converted to an adduct in which cystamine is joined to the 5'-terminal phosphate of the oligonucleotide by a phosphoramidate bond. The adducts are mixed and reduced with dithiothreitol. The dithiothreitol is then removed by dialysis. Oxidation by atmospheric oxygen occurs to yield the required dimer. To join an oligonucleotide to a cysteine-containing peptide or protein, the 5'-cystamine oligomer is first converted to a 2'-pyridyldisulfide adduct and then reacted with an excess of the peptide or protein. If the peptide does not contain a free cysteine residue, it is first treated with iminothiolane to introduce one or more sulfhydryl groups. We have used these procedures to join a 16 mer deoxynucleotide probe and MDV-1 RNA, a substrate of Q beta RNA polymerase. This adduct hybridizes with a complementary target DNA. We have also joined a 16mer probe to peroxidase and MDV-1 RNA to human IgG. The probe-peroxidase adduct maintains enzymatic activity and the MDV-1 RNA-IgG adduct binds to a complementary anti-IgG.