Oxadiazole carboxamide nucleosides as probes for DNA polymerases

Olga V Adelfinskaya, Purdue University


Azole carboxamide nucleosides represent a unique class of analogs that possess distinct electronic properties and structural versatility through the rotation of glycosidic and carboxamide bonds. Therefore, these nucleoside analogs can serve to probe various interactions within an active site of a DNA polymerase in order to understand the mechanics of DNA polymerase replication and fidelity. Structure-activity relationship suggested that contacts between the enzyme and the minor groove of a DNA duplex can be essential for incorporation and extension by a DNA polymerase. Electronic effects of azole nucleobases in DNA polymerase recognition were further explored with a series of oxadiazole carboxamide nucleosides. These C-nucleoside mimics have oxygen and nitrogen atoms at different positions of the heterocycle providing more tunable electrostatic contacts for DNA polymerase recognition and specificity. Conversion of oxadiazole nucleosides to triphosphates was successfully accomplished through enzymatic phosphorylation of oxadiazole diphosphates with nucleoside diphosphate kinase (NDPK). Our observations indicated that oxadiazole carboxamide dNTPs are incorporated by Taq DNA polymerase across from G and T nucleobases whereas Therminator DNA polymerase efficiently inserts these triphosphates opposite to all four natural bases. It was found that both Taq and Therminator DNA polymerases support the primer strand extension and DNA synthesis beyond the oxadiazole nucleobase. Due to incompatibility of oxadiazole carboxamide nucleosides with phosphoramidite synthesis, a method that utilized a solid support for the DNA polymerase mediated extension of an oligodeoxyribonucleotide strand with a modified nucleoside triphosphate was developed. This strategy involved grafting of derivatized latex beads with oligonucleotides and a non-covalent assembly of a primer/template duplex. The extension of the immobilized DNA duplex oxadiazole carboxamide nucleotides was successfully accomplished with several DNA polymerases.




Bergstrom, Purdue University.

Subject Area

Biochemistry|Organic chemistry

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