SITE-SPECIFIC LABELING OF DNA-SEQUENCES CONTAINING PHOSPHOROTHIOATE DIESTERS

A phosphorothioate diester can be incorporated site-specifically into DNA sequences to provide a nucleophilic site that is amenable to alkylation by labels containing haloacetamide, aziridine sulfonamide, or gamma-bromo-alpha,beta-unsaturated carbonyl functionalities. Labeling reactions proceed most efficiently at 50-degrees-C in the pH range 5.0-8.0 and require a number of hours for completion. HPLC techniques employing reversed-phase columns can be used to rapidly purify the labeled materials, and a variety of examples are shown including the purification of a site-specifically labeled 30-mer. Sequences containing a single diastereomeric phosphorothioate (R(p) or S(p)) can be prepared by synthesizing the appropriate dNp(s)N dimer block followed by resolution of the diastereomers and incorporation of either the S(p) or R(p) dimer into the sequence of interest. Oligodeoxynucleotides labeled in this manner are quite stable near neutral pH values, but the phosphorothioate triesters formed undergo hydrolysis under alkaline conditions. Double-stranded sequences containing the labeled phosphorothioate are less prone to base catalyzed hydrolysis than single-stranded sequences. Duplex structures containing a single backbone label are shown to have thermal stabilities that are generally very similar to those of the unlabeled sequences suggesting that little structural perturbation is present for sequences labeled by this technique. Labeling internucleotidic phosphorothioate diesters provides a rapid and simple procedure for the introduction of fluorophores, spin labels, or other moieties site-specifically without significant changes in standard phosphoramidite DNA synthesis techniques.