The preparation of poly (dT)-5'-transferrin conjugates and hybridisation studies with poly (dA)-tailed linearised pBR322 plasmid DNA

The formation of transferrin-DNA complexes intended for ligand-directed transfection studies has been achieved through a hybridisation technique involving complementary homodeoxypolynucleotide chains attached to the participating protein and DNA species. Oligothymidylate residues (pT)n obtained by dicyclohexylcarbodiimide (CDI) polymerisation of thymidine-5'-monophosphate (5'-TMP) were activated to the 5'-imidazolides which on incubation with transferrin yielded the 5'linked phosphoramidates (pT)n-5'-transferrin. Homopolymeric chain extension of (pT)5-5'-transferrin by terminal transferase and dTTP at 30 degrees for 30 min yielded (pT) 300-5'-transferrin. Cleavage of the phosphoramide link in the polymer modified transferrin at 37 degrees was pronounced after 30 min although at 25 degrees hydrolysis was less than 5% after 4 hr. Poly(dT)-5'-transferrin readily hybridised with [3H]poly(dA)-tailed Pst 1 linearised pBR322 DNA. Resultant complexes were demonstrated by nitrocellulose filter binding and immunoprecipitation with anti-transferrin antibody. In contrast with poly(dT)-5'-transferrin, poly(dT)-5'-transferrin-poly(dA)-tailed pBR322 DNA complexes were stable at 37 degrees suggesting that annealing is followed by further stabilising interactions between the DNA and protein components.