The Construction And Application Of Demethylase-Activated Deoxyribozyme

Demethylase-involved removal of N6-methyladenine(m~6A)represents one of the vital epigenetic events,m~6A demethylase is an important epigenetic protease,plays a vital role in the biological prosesses of diseases and has attracted increasing attentions,making the sensitive detection of m~6A demethylases of great significance in clinical disease diagnosis and treatment,yet its direct in stiu evaluation is extremely rare due to the lack of a sensitive and simple detection tool.The endonuclease-mimicking DNAzymes are catalytically active DNAs which have been widely used in the field of biosensor and clinical diagnosis,and several strategies have imparted the magnificent responsiveness to DNAzyme by using chemical and light stimuli.However,the important endogenous biomacromolecules regulation of DNAzyme has remained largely unexplored,leaving a significant gap in responsive DNAzyme regulation.In view of this,a demethylase regulated DNAzyme was developed in this paper for the detection of intracellular demethylase.The main research works have been summarized as follows:(1)This thesis reported a self-responsive DNAzyme system through the in vitro selection of an exquisite m~6A-caged DNAzyme that could be specifically activated by FTO demethylation for precise intracellular FTO imaging.Based on a systematic investigation,the active DNAzyme configuration was potently disrupted by the site-specific incorporation of m~6A modification,and was subsequently restored into the intact DNAzyme structure via the tunable FTO-specific removal of m~6A-caging groups.This demethylase-activated DNAzyme amplifier is able to cleave the double-labelled(fluorophore/quencher pair)substrate to produce a fluorescent signal,enables the robust and accurate monitoring of FTO and its inhibitors in live cells.Moreover,the simple demethylase-activated DNAzyme facilitates the assembly of an intelligent self-adaptive gene regulation platform for knocking down demethylase with an ultimate tumor cells apoptosis.As a straightforward and scarless m~6A removal strategy,the demethylase-activated DNAzyme system offers a versatile toolbox for more different nucleic acid-(de)modifying biotransformations and these relevant enzymes/substrates.(2)To further expand the amplification detection of demethylase and realize accurate estimate of tumor cells.A nanomachine in response to co-stimulation of FTO protein and microRNA was constructed to achieve accurate identification of tumor cells.The DNAzyme was caged by an m~6A group,and blocked by a complementary hybridized DNA.Only in the presence of microRNA,a toehold mediated strand displacement reaction occurs to release the single-stranded m~6A-caged DNAzyme,which could be specifically demethylated by FTO protein to restore the initial DNAzyme structure with high activity,and could cleave the substrate labeled in Au NPs to produce fluorescence signal.Compared with the traditional assay of single biomarker,this design reduces the positive background signal and improves the accuracy of tumor diagnosis by two biomarkers.The combination of entropy driven strand displacement reaction and DNAzyme dual signal amplification,and the confining of nucleic acid probe on the surface of Au NPs can greatly accelerate the reaction rate and improve the detection sensitivity.Realizing a high sensitivity and specificity of intracellular microRNA imaging,and cancer cells could be distinguished according to the differences expression of microRNA and FTO protein.Most importantly,this work fills a gap in the current efficient detection of intracellular FTO proteins,which provided a new insight for simultaneous imaging of multiple biological macromolecules for accurate tumor diagnosis.