7-Deazapurine containing DNA: efficiency of c7GdTP, c7AdTP and c7ldTP incorporation during PCR amplification and protection from endodeoxyribonuclease hydrolysis

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Abstract
The enzymatic synthesis of 7-deazapurine nucleoside containing DNA (501 bp) is performed by PCR-amplification (Taq polymerase) using a pUC18 plasmid DNA as template and the triphosphates of 7-deaza-2'-deoxyguanosine (c 7 G d ), -adenosine (c 7 A d ) and -inosine (c 7 l d ). c 7 G d TP can fully replace dGTP resulting in a completely modified DNA-fragment of defined size and sequence. The other two 7-deazapurine triphosphates (c 7 A d TP) and (c 7 I d TP) require the presence of the parent purine 2'-deoxyribonucleotides. In purine/7-deazapurine nucleotide mixtures Taq polymerase prefers purine over 7-deazapurine nucleotides but accepts c 7 G d TP much better than c 7 A d TP or c 7 l d TP. As incorporation of 7-deazapurine nucleotides represents a modification of the major groove of DNA it can be used to probe DNA/protein interaction. Regioselective phospho-diester hydrolysis of the modified DNA-fragments was studied with 28 endodeoxyribonucleases. c 7 G d is able to protect the DNA from the phosphodiester hydrolysis in more than 20 cases, only a few enzymes (Mae lll, lRsa l, Hind lll, Pvu ll or Taq l) do still hydrolyze the modified DNA. c 7 A d protects DNA less efficiency, as this DNA could only be modified in part. The absence of N-7 as potential binding position or a geometric distortion of the recognition duplex caused by the 7-deazapurine base can account for protection of hydrolysis.