DNA Polymerase Catalyzes Successive Formation of Cytosine-Ag^I-Thymine Base Pairs

Abstract

To explore the constraints of forming consecutive C-Ag^I-T base pairs during DNA polymerase-catalyzed primer extension reactions, we examined the incorporation of deoxythymidine triphosphate (dTTP) into consecutive C sites in the presence of Ag^I ions. The results demonstrated that Ag^I-promoted primer extension enables the formation of up to three consecutive C-Ag^I-T base pairs. Importantly, suppression of C-Ag^I-C base pair formation resulting from dCTP incorporation was demonstrated to be essential for achieving this outcome. Furthermore, by using Mn^II ions, the formation of up to five consecutive C-Ag^I-T base pairs was achieved. The formation of consecutive T-Ag^I-C base pairs, which involves the incorporation of dCTP into templating T sites, is more difficult than the formation of consecutive C-Ag^I-T base pairs, because much higher concentrations of dTTP compete with the template thymine for the Ag^I ion that abstracts the N3 imino proton. Thermal denaturation studies demonstrated that Ag⁺ ions effectively stabilized duplexes containing consecutive C–T mismatches, whereas stabilization by consecutive C-Ag⁺-C base pairs was limited because of electrostatic repulsion between neighboring positively charged base pairs. These results suggest that charge-neutral and thermally stable metal-mediated base pairs are critical for efficient polymerase-catalyzed synthesis of metal-containing DNA. These insights could be beneficial for the enzymatic synthesis of DNA-based Ag^I nanowires.