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 different closing fragments, nick positions, and nucleotides facing the nick were investigated. T4, T5, GTA4C, GCGA2GC, and GCGA3GC sequences were chosen as the closing fragments. The nicks were placed in the center of the duplex stem or were adjacent to the closing fragments. N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide and cyanogen bromide were used as the condensing agents. We showed that the ligation efficiency is 10%-90% depending on the sequence of the closing fragments, nick position, and nucleotides facing the nick. Coupling yields of 80%-90% were observed when the nick was situated in the middle of the molecule between two T residues or was adjacent to GCGA2GC or GCGA3GC minihairpins. In the last case, the reacting 3'-phosphate and 5'-hydroxy groups were brought close together by only two base pair minihairpins. The coupling yields did not depend on the nature of the condensing agent. On the basis of the results obtained, we believe a rational design of nicked DNA dumbbells has been developed for efficient chemical synthesis of closed dumbbells.
Kuznetsova
S A
SA
Department of Chemistry, Moscow State University, Russia.
Merenkova
I N
IN
Kanevsky
I E
IE
Shabarova
Z A
ZA
Blumenfeld
M
M
D020029:Base Pairing; D001483:Base Sequence; D003488:Cyanogen Bromide / Q000737:chemistry; D004247:DNA / Q000138:chemical synthesis* / Q000737:chemistry / Q000235:genetics; D004268:DNA-Binding Proteins; D015195:Drug Design; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000737:chemistry; D051537:Hepatocyte Nuclear Factor 1; D051539:Hepatocyte Nuclear Factor 1-beta; D007700:Kinetics; D009687:Nuclear Proteins; D009690:Nucleic Acid Conformation; D011089:Polydeoxyribonucleotides / Q000737:chemistry* / Q000235:genetics; D020218:Response Elements / Q000235:genetics; D013698:Templates, Genetic; D013816:Thermodynamics; D014157:Transcription Factors / Q000378:metabolism
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D011089:Polydeoxyribonucleotides; D014157:Transcription Factors; D051537:Hepatocyte Nuclear Factor 1; D051539:Hepatocyte Nuclear Factor 1-beta; D004247:DNA; D003488:Cyanogen Bromide; D005022:Ethyldimethylaminopropyl Carbodiimide