2-Substituted 2'-deoxyinosine 5'-triphosphates as substrates for polymerase synthesis of minor-groove-modified DNA and effects on restriction endonuclease cleavage
Abstrakt
Five 2-substituted 2'-deoxyinosine triphosphates (d (R) ITP) were synthesized and tested as substrates in enzymatic synthesis of minor-groove base-modified DNA. Only 2-methyl and 2-vinyl derivatives proved to be good substrates for Therminator DNA polymerase, whilst all other d (R) ITPs and other tested DNA polymerases did not give full length products in primer extension.
The DNA containing 2-vinylhypoxanthine was then further modified through thiol-ene reactions with thiols. Cross-linking reaction between cysteine-containing minor-groove binding dodecapeptide and DNA proceeded thanks to the proximity effect between thiol and vinyl groups inside the minor groove. 2-Substituted dI (R) TPs and also previously prepared 2-substituted 2'-deoxyadenosine triphosphates (d (R) ATP) were then used for enzymatic synthesis of minor-groove modified DNA to study the effect of minor-groove modifications on cleavage of DNA by type II restriction endonucleases (REs).
Although the REs should recognize the sequence through H-bonds in the major groove, some minor-groove modifications also had an inhibiting effect on the cleavage.