Construction Of Fluorescent Biosensors For Demethylase Assay

DNA methylation and RNA methylation can induce heritable,reversible changes in gene expression without changing the sequences of nucleic acids.They are directly involved in various biological processes including gene expression,cell development,and chromosome inactivation,playing important roles in the maintenance of genomic integrity.DNA demethylation and RNA demethylation can maintain the dynamic equilibrium process of DNA and RNA methylation,and the dysregulation of the balance can affect normal gene activation and expression.Therefore,demethylases are essential for maintaining the integrity of DNA and RNA.The common DNA demethylases include: methylated Cp G binding domain(MBD)family proteins,TET(Ten-eleven translocation)family proteins and methylation transferase(DNMT)family proteins,etc.RNA demethylases are mainly Alk B(ALKBH1-8 and FTO)family proteins.Recent researches demonstrate that the aberrant activity of DNA and RNA demethylases may cause a variety of human diseases(e.g.,neurological damage,Alzheimer’s disease and hematopoietic disorders)and cancers(e.g.,breast,liver,lung cancer).Therefore,the sensitive detection of demethylases is great importance for biological research,disease diagnosis and drug discovery.Traditional methods for detection of demethylases include western blotting,enzyme linked immunosorbent assay(ELISA),chromatin immunoprecipitation assay(Ch IP),classic thin-layer chromatography(TLC),liquid chromatography-mass spectrometry(LC-MS).However,most of these methods are severely limited due to the involvement of a large quantity of specific antibodies,time-consuming separation procedures,large sample injection,and poor sensitivity.Therefore,there is an urgent need to develop rapid,sensitive and simple methods for the detection of demethylases activity.Therefore,sensitive detection of demethylase is significant in biological research,disease diagnosis,and drug discovery.Western blotting,enzyme linked immunosorbent assay,chromatin immunoprecipitation assay,classic thin-layer chromatography,liquid chromatography-mass spectrometry,fluorescence polarization measurement,and electrochemical assay are frequently used for demethylase assay.However,most of them involve a large quantity of specific antibodies time-consuming separation procedures large sample-injection,and poor sensitivity.Alternatively,a variety of efficient amplification strategies have been employed to improve the sensitivity,but they usually require complexes probes/templates design and exhibit high background signal arising from nonspecific amplification.Therefore,sensitive and simple methods for demethylase assay are highly required.In this thesis,two kinds of fluorescence biosensors were developed for the detection of demethylases with TET2,FTO and ALKBH3 serve as research targets.This thesis includes the following contents:1.Development of a single quantum dot-mediated FRET biosensor for amplification-free detection of ten-eleven translocation 2We develop a single-quantum-dot(QD)-mediated fluorescence resonance energy transfer(FRET)biosensor for amplification-free measurement of teneleven translocation 2(TET2).In this work,when TET2 is present,it catalyzes the oxidation of 5-vinylcytosine in ds DNA to 5-formylmethylcytosine,and the subsequent labeling of ds DNA with Cy5 generates a biotinylated Cy5-ds DNA complex.Biotinylated Cy5-ds DNA complexes are conjugated to the streptavidincoated 605 QDs to obtain a Cy5-ds DNA-605 QD nanostructures,inducing FRET from 605 QD to Cy5.FRET signal can be simply measured by single-molecule counting.This biosensor enables homogeneous detection of TET2 with a limit of detection(LOD)of 0.042 ng/μL,and it can accurately measure cellular TET2 down to 1 cell.Moreover,this biosensor can be used to screen TET2 inhibitors,offering a new platform for TET2-related medical research and clinical diagnostics.2.Single-molecule detection of FTO and ALKBH3 based on ligation cyclic amplificationWe developed a sensitive method for simultaneous detection of FTO and ALKBH3 based on the integration of ligase amplification reaction with singlemolecule detection.In this work,when FTO is present,m6 A is converted to A,generating a demethylated probe 1.Then demethylated probe 1 enables hybridization with capture probe 1 and 2 to initiate ligase amplification reaction by recruiting Taq DNA ligase and generating long strand DNA.The resultant longstranded DNA can hybridize with signal probe 1 and 2 and continue to initiate the ligase amplification reaction by using Taq DNA ligase,producing biotin-Cy3 duallabeled signal probe.Similarly,ALKBH3 can catalyze the demethylation of m1 A,producing a demethylated probe 2.Then demethylated probe 1 enables hybridization with capture probe 1 and 3 to initiate ligase amplification reaction by recruiting Taq DNA ligase and generating long strand DNA.The resultant longstranded DNA can hybridize with signal probe 1 and 3 and continue to initiate the ligase amplification reaction by using Taq DNA ligase,producing biotin-Cy5 duallabeled signal probe.Biotin-Cy3 dual-labeled signal probes and biotin-Cy5 duallabeled signal probes can assemble on the surface of streptavidin-coated magnetic beads(MBs)to form MB-single stranded DNA-Cy3/Cy5 nanostructure.After DNase I digestion and magnetic separation steps,Cy3 and Cy5 fluorescent molecules are released into solution and subsequently counted by total internal reflection fluorescence(TIRF)-based single molecule detection,with the Cy3 signals indicating the presence of FTO and the Cy5 signals indicating the presence of ALKBH3.This method exhibits good specificity and high sensitivity detection limit of 3.82×10-10 ng/μL(equivalent to 6.33×10-18 mol/L)for FTO and 5.33×10-10ng/μL(equivalent to 1.44×10-17 mol/L)for ALKBH3.Importantly,it can even quantify FTO and ALKBH3 activities at the single-cell level,with great potential in FTO and ALKBH3-related biomedical research and disease diagnosis.