Active DNA demethylation, mediated by O-2-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-2 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-2 in a wide range from cellular hypoxia (<= 15 mu M) to ambient condition (similar to 257 mu M). By further immobilization of the MOFs onto transparent silicon rubber (MOF@SR) to construct O-2 film sensors, and real-time monitoring of O-2 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-m) values of TET2 for 5mC and O-2 in ascorbic acid-free (AA(-)) condition were precisely evaluated to be 24 +/- 1 and 43.8 +/- 0.3 mu M, 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-2, Fe2+) 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-2-related enzymes.
[1]Sun Yat Sen Univ, Sch Chem, Guangzhou 510275, Guangdong, Peoples R China;
[2]Fudan Univ, Shanghai Canc Ctr, Inst Biomed Sci, Shanghai Med Coll, Shanghai 200032, Peoples R China;
[3]Fudan Univ, Sch Life Sci, State Key Lab Genet Engn, Shanghai 200433, Peoples R China
(8.0+极速模式)