Efficient synthesis of DNA dumbbells using template-induced chemical ligation in double-stranded polynucleotides closed by minihairpin fragments

The chemical ligation of 17 50-54-membered nicked DNA dumbbells with differ ent closing fragments, nick positions, and nucleotides facing the nick were investigated. T-4, T-5, GTA(4)C, GCGA(2)GC, and GCGA(3)GC sequences were c hosen as the closing fragments. The nicks were placed in the center of the duplex stem or were adjacent to the closing fragments. N-(3-dimethylaminopr opyl)-N '-ethylcarbodiimide and cyanogen bromide were used as the condensin g agents. We showed that the ligation efficiency is 10%-90% depending on th e sequence of the closing fragments, nick position, and nucleotides facing the nick, Coupling yields of 80%-90% were observed when the nick was situat ed in the middle of the molecule between two T residues or was adjacent to GCGA(2)GC or GCGA(3)GC minihairpins, In the last case, the reacting 3'-phos phate and 5'-hydroxy groups were brought close together by only two base pa ir minihairpins, The coupling yields did not depend on the nature of the co ndensing agent. On the basis of the results obtained, we believe a rational design of nicked DNA dumbbells has been developed for efficient chemical s ynthesis of closed dumbbells.