Structural basis of the phosphoramidate N-benzimidazole group’s influence on modified primer extension efficiency by Taq DNA polymerase

We recently proposed a novel class of nucleic acid derivatives - phosphoramidate benzoazole oligonucleotides (PABAOs). In these compounds, one of the non-bridging oxygen atoms is replaced by a phosphoramidate N-benzoazole group, such as benzimidazole, dimethylbenzimidazole, benzoxazole, or benzothiazole. Studies of the properties of these derivatives have shown that their use in PCR enhances the specificity and selectivity of the analysis. The study investigates the effect of phosphoramide N-benzimidazole modification of DNA primers on their elongation by Taq DNA polymerase using molecular dynamics simulations. We examined perfectly matched primer-template complexes with modifications at positions one through six from the 3'-end of the primer. Prior experimental work demonstrated that the degree of elongation suppression depends on the modification position: the closer to the 3'-end, the stronger the inhibition, with maximal suppression observed for the first position, especially in mismatched complexes. Furthermore, incomplete elongation products were experimentally observed for primers modified at the fourth position. Our molecular dynamics simulations and subsequent analysis revealed the molecular mechanisms underlying the interaction of modified primers with the enzyme. These include steric hindrance that impedes polymerase progression along the modified strand and local distortions in the DNA structure, which explain the experimentally observed trends. We established that both different stereoisomers of the phosphoramidate groups and conformers of the phosphoramidate N-benzimidazole moiety differentially affect the structure of the enzyme-substrate complex and the efficiency of Taq DNA polymerase interaction with the modified DNA complex. Modification of the first and second internucleoside phosphate from the 3'-end of the primer causes the most significant perturbation to the structure of the protein-nucleic acid complex. When the modification is located at the fourth phosphate group, the N-benzimidazole moiety occupies a specific pocket of the enzyme. These findings provide a foundation for the rational design of specific DNA primers bearing modified N-benzimidazole moieties with tailored properties for use in PCR diagnostics.