Electrochemistry of epigenetic modulator zebularine: Voltammetric determination in cell culturing media and monitoring of polymerase incorporation into DNA

Abstract

Electrochemical properties of epigenetic modulator zebularine (Zeb) were studied for the first time using cyclic voltammetry (CV) at three types of working electrodes (hanging mercury drop electrode – HMDE; mercury meniscus-modified silver solid amalgam electrode - m-AgSAE; and basal plane pyrolytic graphite electrode – bPGE). CV responses of Zeb were investigated within the pH range from 2 to 12. Depending on the electrode type, Zeb gave up to three cathodic peaks (“c1”, “c2”, “c3”) and an anodic peak “a” corresponding to oxidation of the product of Zeb reduction. Using optimized pretreatment of the solid electrodes, concentration dependences of the peak c1 were measured in ammonium formate, phosphate buffer pH 6.9 at the m-AgSAE, or in sodium acetate buffer pH 5.0 at the bPGE. LOD of 5 µM was achieved with all three electrodes in the buffer solutions. Using HMDE, the same LOD was attained also in cell culturing media without any sample pretreatment. Incorporation of 2′-deoxyzebularine 5′-triphosphate into DNA was studied using primer extension and terminal deoxynucleotidyl transferase tailing techniques. Incorporation of Zeb exhibited poor efficacy; nevertheless, products of TdT tailing with deoxyZeb were successfully detected by ex situ CV. Interestingly, another epigenetic modulator, 5-azacytidine showed facile incorporation in both approaches. These observations were in accord with the presumably different mechanism of action of the two epigenetic drugs in cells. We demonstrate usefulness of a simple electrochemical method to assess different facileness of polymerase incorporation of two epigenetic drugs, cytidine analogues Zeb and 5-aC, by DNA polymerases.