A DNA nanoscaffold-based electrochemical assay for sensitive determination of O-GlcNAc transferase (OGT) activity and its application in cell-permeable OGT inhibitors screening

Abstract

Human O-GlcNAc transferase (OGT) is responsible for regulating O-GlcNAc glycosylation and has emerged as a potential therapeutic target. Abnormal regulation of OGT is usually related to the pathogenesis of diabetes and cancer. Therefore, the quantification of OGT activity is necessary for drug discovery and clinical diagnosis. To improve the accuracy and sensitivity of electrochemical analysis for OGT activity, we herein design an OGT-responsive peptide to couple with tetrahedral DNA nanostructure (TDN) scaffold on a gold electrode. The peptide includes an O-GlcNAcylated site, a protease recognition site, and a biotinylated end for electric signal amplification. The TDN shows good stability in the high level of nuclease and the cell lysate. With the OGT, the streptavidin-conjugated horseradish peroxidase (HRP) can connect with the peptide to catalyze the electric substrate. Based on the current response, we can detect the OGT activity with higher sensitivity (LOD = 0.153 nM) than coupling flexible DNA on the electrode. Then, the sensing system is successfully used for OGT detection in cell lysate without enzyme enrichment. Finally, we apply this assay to detect the inhibition effect of OGT inhibitor OSMI-1 in HEK293 cells (IC50 = 10.34 μM), providing important information to evaluate the intracellular inhibition efficiency of OGT inhibitors. In summary, the proposed electrochemical biosensor could be promisingly used in a convenient miniaturized device to screen cell-permeable OGT inhibitors.