Research On Ultrasensitive Detection Of Demethylase FTO Based On Single Quantum Dot Nanosensor

Nucleic acid modifications exist in various life forms and plays a vital role in the regulation of gene expression.Till now,over 100 different kinds of post-transcriptional modifications found in RNA.Among these RNA modifications,N⁶-methyladenosine?m⁶A?is the most common base modification in eukaryotic mRNA,and more than 80%of RNA bases are methylated,and there is 1 m⁶A per 2000 ribonucleotides on average.The m⁶A is highly involved in post-transcriptional regulation,such as mRNA splicing,translation,nuclear export,mRNA attenuation,subcellular localization,and tissue-specific regulation,and the disorders of m⁶A modifications are closely related to a variety of human diseases.Recent resaerches show that the fat mass and obesity-associated FTO enzyme play an important role in the dynamic regulation of cellular levels of m⁶A modification,and the FTO enzyme can catalyze the demethylation of N⁶-methyladenosine residues in mRNA and regulates the cellular level of m⁶A modification,playing a critical role in human obesity and cancers treatment.In view of the important role of FTO as m⁶A demethylase in obesity and other diseases,the highly sensitive detection of FTO activity and the screening of small molecule inhibitors are of great significance in biological research,clinical diagnosis and drug development.Quantum dots are a new type of semiconductor nanomaterial,in comparison with traditional dye molecules,quantum dots have a variety of advantages,and special optical properties make them have wide applications in biochemistry,molecular biology,cell biology,and biological macromolecular interaction.In this thesis,we developed a quantum dots based fluorescence resonance energy transfer?FRET?sensor to sensitively detect demethylase FTO activity and screen its potential inhibitors.This assay consists of three consecutive steps:?1?FTO-mediated demethylation against the single-strand methylated DNA substrate,?2?the Dpn II endonuclease-assisted cleavage of double-stranded demethylated DNA substrate to generate biotinylated capture probe,?3?the construction of QD-DNA-Cy5 nanostructure and detection of fluorescence resonance energy transfer signal.First,FTO-mediated demethylation of m⁶A can induce Dpn II to specifically cleave demethylated DNA for the production of biotinylated DNA fragments.Then this DNA fragment acts as a capture probe to assemble a Cy5-labeled reporter probe onto the surface of a quantum dot to trigger the fluorescence resonance energy transfer between quantum dot and Cy5,and the resulting Cy5 signal can be monitored by total internal reflection fluorescence?TIRF?imaging system.By simply counting the Cy5 molecules,FTO activity can be accurately quantified.This assay is very simple and fast,without the requirement of any external amplification,any separation or washing steps.The limit of detection is as low as 7.9?10^-14 M.In addition,we screened FTO-related inhibitors.The presence of inhibitors can inhibit FTO demethylation,thereby inhibiting the fluorescence resonance energy transfer between quantum dot and Cy5.The inhibitory effect of inhibitors upon FTO demethylation can be easily assessed by monitoring the decrease of Cy5 signal.We used this nanosensor to screen several small molecule inhibitors and identified Diacerein as a highly selective FTO inhibitor.Diacerein can inhibit the demethylation of endogenous FTO in HeLa cells.Diacerein is neither a structural analog of 2-oxoglutarate?2-OG?nor a chelator of metal ions,and it can selectively inhibit FTO demethylation by competitively binding the m⁶A-containing substrate.