Site-directed modification of DNA duplexes by chemical ligation

• Published in: Nucleic acids research Vol. 16; no. 9; pp. 3721 - 3738
• Main Authors: DOLINNAYA, N. G, SOKOLOVA, N. I, GRYAZNOVA, O. I, SHABAROVA, Z. A
• Format: Journal Article
• Language: English
• Published: Oxford Oxford University Press 11-05-1988
• Subjects: Analytical, structural and metabolic biochemistry; Base Composition; Base Sequence; Biological and medical sciences; DNA; Dna, deoxyribonucleoproteins; Electrophoresis, Polyacrylamide Gel; Fundamental and applied biological sciences. Psychology; Nucleic Acid Conformation; Nucleic acids; Oligodeoxyribonucleotides - chemical synthesis; Phosphorus Radioisotopes; Phosphorylation; Structure modification; Method; Chemical bond; DNA; Duplex
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/16.9.3721

The efficiency of chemical ligation method have been demonstrated by assembling a number of DNA duplexes with modified sugar phosphate backbone. Condensation on a tetradecanucleotide template of hexa(penta)- and undecanucleotides differing only in the terminal nucleoside residue have been performed using water-soluble carbodiimide as a condensing agent. As was shown by comparing the efficiency of chemical ligation of single-strand breaks in those duplexes, the reaction rate rises 70 or 45 times if the 3'-OH group is substituted with an amino or phosphate group (the yield of products with a phosphoramidate or pyrophosphate bond is 96-100% in 6 d). Changes in the conformation of reacting groups caused by mismatched base pairs (A.A, A.C) as well as the hybrid rU.dA pair or an unpaired base make the template-directed condensation less effective. The thermal stability of DNA duplexes was assayed before and after the chemical ligation. Among all of the modified duplexes, only the duplex containing 3'-rU in the nick was found to be a substrate of T4 DNA ligase.