Real-Time Sensing of TET2-Mediated DNA Demethylation In Vitro by Metal-Organic Framework-Based Oxygen Sensor for Mechanism Analysis and Stem-Cell Behavior Prediction.

Active DNA demethylation, mediated by O-dependent ten-eleven translocation (TET) enzymes, has essential roles in regulating gene expression. TET kinetics assay is vital for revealing mechanisms of demethylation process. Here, by a metal-organic framework (MOF)-based optical O sensor, we present the first demonstration on real-time TET2 kinetics assay in vitro. A series of luminescent Cu(I) dialkyl-1,2,4-triazolate MOFs were synthesized, which were noble-metal-free and able to intuitively response to dissolved O in a wide range from cellular hypoxia (≤15 μM) to ambient condition (∼257 μM). By further immobilization of the MOFs onto transparent silicon rubber (MOF@SR) to construct O film sensors, and real-time monitoring of O consumption on MOF@SR over the reaction time, the complete TET2-mediated 5-methylcytosine (5mC) oxidation process were achieved. The method overcomes the limitations of the current off-line methods by considerably shortening the analytical time from 0.5-18 h to 10 min, and remarkably reducing the relative standard deviation from 10%-68% to 0.68%-4.2%. As a result, the Michaelis-Menten constant ( K) values of TET2 for 5mC and O in ascorbic acid-free (AA) condition were precisely evaluated to be 24 ± 1 and 43.8 ± 0.3 μM, respectively. By comparative study on AA-containing (AA) conditions, and further establishing kinetics models, the stem-cell behavior of TETs was successfully predicted, and the effects of key factors (AA, O, Fe) on TETs were revealed, which were fully verified in mouse embryonic stem (mES) cells. The method is promising in wide application in kinetics analysis and cell behavior prediction of other important O-related enzymes.