2-Substituted 2′-deoxyinosine 5′-triphosphates as substrates for polymerase synthesis of minor-groove-modified DNA and effects on restriction endonuclease cleavage

Abstract

Five 2-substituted 2′-deoxyinosine triphosphates (d^RITP) 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^RITPs 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^RTPs and also previously prepared 2-substituted 2′-deoxyadenosine triphosphates (d^RATP) 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.