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Nucleic Acids Research, 1988, Vol. 16, No. 9 3721-3738
© 1988

Articles

Site-directed modification of DNA duplexes by chemical ligation

N.G. Dolinnaya, N.I. Sokolova, O.I. Gryaznova and Z.A. Shabarova

The Belozersky Laboratory of Molecular Biology and Bioorganic Chemistry and Chemical Department, Moscow State University Moscow 119899, USSR

Received February 9, 1988. Accepted March 8, 1988.

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.


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R. C. Alexander, A. K. Johnson, J. A. Thorpe, T. Gevedon, and S. M. Testa
Canonical nucleosides can be utilized by T4 DNA ligase as universal template bases at ligation junctions
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