Enzymatic ligation of synthetic DNAs carrying point amidophosphate internucleotide linkage modification
E. V. Shybanova, S. A. Filippov, D. S. Esipov, V. G. Korobko, V. N. Dobrynin
M. M. Shemyakin Institute of Bioorganic Chemistry, Academy of Sciences of the USSR, Moscow
Abstract: DNA fragments with the point amidophosphate (cyclohexylamido- or morpholido-) modification in the sugar-phosphate backbone were synthesized and separated into individual diastereoisomer. The isomers were separated by the reversed-phase HPLC (RFC), and chirality at phosphorus was assigned by a stcreochemical correlation scheme using phosphorothioate standards. The RFC-retention time values for Rp -isomers were found to be lower than for Sp-analogues. Amidophosphate DNA fragments were used as P-and OH-compononts in the T4 DNA-ligation. The enzyme does not ligate amidated fragments with modified internucleotide linkage near 5'- or 3'-end, independently of the ami-dophosphate chirality. When an unmodified phosphodiester linkage separates the amidophosphate group from 3'-end in O-component, the ligation occurs only with Sp -isomer, whereas fly-analogue does not give the ligation product. In the P-component of the ligation, configuration of the modified linkage separated from 5'-phosphato by an unmodified linkage does not affect the result of the enzymatic reaction: both Sp-and Rp-stereo-mers do take part in the ligation. As a result of the ligation of the modified fragments on unmodified templates a set of 31-mers was obtained. They contain FokI and EcoRI recognition sites with the cleavage points of both endonucleases coinciding and being amidated. Upon treatment of duplex DNA consisted of unmodified and amidated strands with those endonucleases Sp-configuration did not hinder the cleavage of the unmodified strand, whereas Rp-configuration inhibited the EcoRI and did not affect the FokI cleavage.
Russian Journal of Bioorganic Chemistry 1991, 17 (1):99-106