[Synthesis and properties of covalently-closed DNA duplexes containing pyrophosphate and and substituted pyrophosphate internucleotide groups]

Abstract

A new method for the efficient synthesis of covalently closed DNA duplexes (DNA dumbbells) and the introduction of pyrophosphate and substituted pyrophosphate internucleotide groups into their structure is proposed. The method is based on chemical ligation in DNA duplexes that are formed by a polynucleotide the ends of which are brought together due to the introduction of the minihairpin structure [sequence: see text]. DNA dumbbells containing a pyrophosphate (substituted pyrophosphate) group result from the interaction as being between the 3'-terminal phosphate (methylphosphate) group of the polynucleotide and the 5'-terminal phosphate group of deoxyguanosine of the minihairpin sequence, which flanks the polynucleotide from the 5' end. 1-Ethyl-3-(3'-dimethylaminopropyl) carbodiimide was used as a condensing agent. The yield of covalently closed 42-mer DNA duplexes containing a pyrophosphate group was 98%, that of duplexes with a substituted pyrophosphate group was 25%. The reactivity of the substituted pyrophosphate group incorporated into DNA dumbbells was studied. It is shown that the group efficiently interacts with nucleophiles in an aqueous medium at pH 8.0.