Novel Sensing Assay For Detection Of Histone Demethylase Activity

Post-translational modification of histones plays an important role in regulating gene expression and remodeling chromatin structure.Histone methylation is one of an important post-translational modification that occurs at the tail of histones.Histone methylation was considered a static and irreversible modification for a long time until the first histone demethylase（lysine specific demethylase 1）was discovered,which reveals the reversible regulation mechanism of histone methylation.Histone demethylase are involved in many biological processes and the abnormal expression of HDMs is related to a variety of diseases.Therefore,analyzing the activity of HDMs is of great significance.However,the tools available to detect HDMs activity remain limited.Since HCHO was produced in the demethylation process of LSD1,we developed several methods for LSD1 activity assay based on the detection of HCHO in this work.The specific research is as follows:1.A dual-readout assay with fluorescence and colorimetric signals was constructed for sensitive detection of HCHO.Ag⁺can catalyze the colorless o-phenylenediamine（OPD）to yellow 2,3-diaminophenazine（OPDox）and generate fluorescence.We found that when HCHO is present in the system,HCHO will hinder the oxidation of OPD by Ag⁺,and cause the color unchanged and no fluorescence generated.Several experiments were carried out to primarily illustrate the mechanism,including changing the order of HCHO addition,the concentration of Ag⁺and OPD,and conducting transmission electron microscopy（TEM）characterization.Accordingly,we speculated that HCHO affects the catalytic ability of silver nanoparticles（Ag NPs）by changing its morphology,thereby hinders the oxidation of OPD by Ag⁺.After conditions optimization,HCHO was sensitively detected through fluorescent and colorimetric dual-signal output,with the response range of 0-60μM,and the detection limit of 0.29μM（fluorescence）and 0.33μM（colorimetric）,respectively.In addition,the assay has good selectivity for HCHO,but not ions and other small molecules.2.Based on the proposed Ag⁺-OPD system,a novel assay was achieved for detecting LSD1 activity.According to the fact that HCHO is generated during the demethylation process catalyzed by LSD1,the fluorescence and colorimetric dual-signal assay for HCHO detection is further used for the detection of LSD1 activity.The response range of the assay to LSD1 is 0-300 nM,and the detection limit is as low as 0.3 nM（fluorescence）and 0.5 nM（colorimetric）,respectively.LSD1 inhibition was also evaluated by the assay with its typical inhibitor,GSK-LSD1,and its IC₅₀ value was 10.23 nM.In addition,this method was also successfully used to detect the LSD1activity in different cell lysates,and it was found that the activity of LSD1 in Hep G2cells is about 2.4 times of that of LO2 cells,which was consistent with the results of Western Blot（WB）and the reports of the literature,which suggests the potential of our method for clinical diagnosis.3.Noval methods for the detection of LSD1 activity was developed based on a small molecule fluorescent probe,N-propyl-4-hydrazinonaphthalimide（Na-FA）.Na-FA was synthesized through a two-step method,and then was purified and characterized by nuclear magnetic.HCHO can specificly"turn on"the fluorescence of Na-FA.The probe is further used for the detection of LSD1.The response range for LSD1 is 5-500 nM and the detection limit is 0.8 nM.In addition,LSD1 activity was successfully detected in He La cell lysate.