High-purity preparation of a large DNA dumbbell

We report on the efficient biochemical synthesis of a large DNA dumbbell st arting from a pair of short DNA hairpins with long single-stranded tails of arbitrary sequence. The DNA dumbbell is obtained by enzymatic ligation yie lding a 94-bp duplex stem closed at both termini by single-stranded loops o f 5 nt, Following ligation, all unligated precursors and monoligated by-pro ducts were multiply biotinylated via nick-translation or primer-extension o r both. Thus, they could readily be removed from the DNA dumbbell preparati on by a mild biomagnetic separation procedure. The closed conformation of t he purified DNA dumbbell was verified by its altered gel mobility as compar ed with unligated or monoligated samples and by an exonuclease assay. Consi dering the promising therapeutic potential of DNA dumbbells, the developed biosynthetic approach could be used for high-purity preparation of longer, covalently closed DNA decoys.