Site-directed modification of DNA duplexes by chemical ligation.
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.
Dolinnaya
N G
NG
Belozersky Laboratory of Molecular Biology and Bioorganic Chemistry, Moscow State University, USSR.
Sokolova
N I
NI
Gryaznova
O I
OI
Shabarova
Z A
ZA
D001482:Base Composition; D001483:Base Sequence; D004247:DNA; D004591:Electrophoresis, Polyacrylamide Gel; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation
D009838:Oligodeoxyribonucleotides; D010761:Phosphorus Radioisotopes; D004247:DNA