[Enzymatic ligation of DNA fragments containing phosphoamide modification of the internucleotide bonds]

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 (RPC), and chirality at phosphorus was assigned by a stereochemical correlation scheme using phosphorothioate standards. The RPC-retention time values for Rp-isomers were found to be lower than for Sp-analogues. Amidophosphate DNA fragments were used as P- and OH-components in the T4 DNA-ligation. The enzyme does not ligate amidated fragments with modified internucleotide linkage near 5'- or 3'-end, independently of the amidophosphate chirality. When an unmodified phosphodiester linkage separates the amidophosphate group from 3'-end in O-component, the ligation occurs only with Sp-isomer, whereas Rp-analogue does not give the ligation product. In the P-component of the ligation, configuration of the modified linkage separated from 5'-phosphate by an unmodified linkage does not affect the result of the enzymatic reaction: both Sp-and Rp-stereomers 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 these 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.