Unnatural imidazopyridopyrimidine:naphthyridine base pairs: selective incorporation and extension reaction by DNA polymerases

Abstract

We describe herein the results of (i) enzymatic recognition for imidazopyridopyrimidine (Im):naphthyridine (Na) base pairs and (ii) further primer extension reactions after the Im:Na base pairs by DNA polymerases. Among the base pairs examined, ImN(O):NaO(N) base pair was rather selectively recognized by Klenow fragment exo(-) [F(exo(-))]as complementary base. However, this DNA polymerase did not catalyze primer extension reactions after the ImN(O):NaO(N) base pair. Therefore, we carried out a screening of DNA polymerases to promote the primer extension reaction as well as to improve the selectivity of base pair recognition. As a result, a family B DNA polymerase, especially Deep Vent (exo(-)), seemed most promising for this purpose.

Mirror-Image Thymidine Discriminates against Incorporation of Deoxyribonucleotide Triphosphate into DNA and Repairs Itself by DNA Polymerases

“…The tip of the thumb domain makes contacts with the primer strand, whereas the architectures of the contacting loops differ greatly: the loop in Therminator structure hoves above the minor groove without extending deeply into it, while the corresponding loop in Taq extends over the phosphate backbone toward the major groove . Furthermore, crystallographic studies have shown a higher-ordered interaction in the minor groove of the double helix DNA with family A polymerases compared to family B polymerases . Because the contacts of all sugars and nucleobases are through the minor groove, l -T nucleotide might clash with the tip of the domain when processed by Taq.…”

“…39 Furthermore, crystallographic studies have shown a higher-ordered interaction in the minor groove of the double helix DNA with family A polymerases compared to family B polymerases. 50 Because the contacts of all sugars and nucleobases are through the minor groove, L-T nucleotide might clash with the tip of the domain when processed by Taq. Thus, Taq shows no tolerance for L-T in the template, whereas better performance of B-family polymerases in processing L-T may be the result of a sterically less-hindered minor groove.…”