Developing High-Performance Fluorogenic Probes for Detecting Lysine Demethylase Activity

Project: Research

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Lysine demethylases play critical roles in regulating many cellular events. Biological studies have shown that malfunction of lysine demethylases is associated with a number of important diseases such as cancers and neurological diseases. Consequently, it has aroused much interest to develop chemical tools for detecting lysine demethylase activity. A number of methods have been developed to detect lysine demethylase activity, including enzyme-coupled approach, antibody-based assays, and others. Nevertheless, few fluorescent probes are available that allow detection of endogenous lysine demethylase activity in native cellular environment.In this proposal, we seek to develop novel fluorogenic probes to detect lysine demethylase activity. In the first stage, we plan to design and synthesize high-performance fluorophores based on rhodamine. By fine tuning the substituents at the spirolactam nitrogen in rhodamine, we aim to develop new fluorophores with high fluorogenicity and cell permeability. After obtaining suitable fluorophores, a HaloTag ligand will be introduced to the fluorophores to examine their fluorescence turn on ability. Cell imaging study will be performed to examine whether the probe is able to image HaloTag protein in non-wash conditions. Furthermore, we will adopt similar approach to develop NIR based fluorophores with high fluorogenicity. NIR fluorophores have shown advantages in bioimaging such as reduced phototoxicity and increased tissue penetration. This will further expand the tool box of fluorogenic probes for live-cell imaging applications.Building on these results, we will move on to design LSD1 specific probes. Specifically we will introduce the optimum fluorophores to the known potent LSD1 inhibitor scaffolds. After verifying the probe’s chemical structure, we will first test the fluorescence response of the probes with recombinant LSD1 in concentration- and time- dependent manners. Subsequently the probes will be applied to imaging LSD1 activity in living cells. SiRNA and inhibition experiments will be performed to examine whether the probes detect LSD1 activity selectively. Furthermore, super-resolution imaging will be conducted to investigate the detailed cellular localization and the movement of LSD1 during DNA damage process. Through the proposed research, we hope to generate robust fluorogenic probes for elucidating the roles of LSD1 in biology. The current research team has extensive experience in fluorescent probe design and the bioimaging field. We are confident that the research will be completed successfully and contribute to the advancement of bioimaging and epigenetic research.


Project number9043206
Grant typeGRF
Effective start/end date1/01/22 → …