Single-molecule Detection Of DNA Glycosylase And RNA Demethylase

Cancer（malignant tumor）is the most important disease threatening human life and health in the world.There are no obvious symptoms in the early stage of cancer,and even if there are symptoms,they are often non-specific.When patients have specific symptoms,they are often in the advanced stage,which poses a great challenge to the early diagnosis and treatment of cancer patients.Therefore,early detection,early diagnosis and treatment of cancer are extremely important to delay the disease and reduce the mortality of patients.Tumor markers are produced when the human body undergoes cancer,and the abnormal expression of tumor markers occurs in the early stage of cancer.However,the abundance of early tumor markers is very low,and traditional detection methods suffer from poor sensitivity,complex operation,time-consuming,and need a large number of samples,which cannot effectively detect specific tumor markers in patients with early cancer.Therefore,there is an urgent need to develop more sensitive and accurate detection methods for tumor markers.Fortunately,single-molecule detection techniques developed in recent years,especially single-molecule fluorescence detection techniques,have the advantages of high sensitivity,high resolution,and easy operation,which provide a new method for the early detection of cancer tumor markers and biomedical research.DNA glycosylases and RNA demethylases are very important tumor markers.Aberrant expression of human 8-oxoguanine DNA glycosylase（h OGG1）may result in various human diseases such as Parkinson’s disease,autoimmune disease,and different types of cancers（e.g.,breast,lung,orolaryngeal,colon,and gastric cancers）.N⁶-methyladenosine RNA demethylase（FTO）is involved in various disease processes such as type II diabetes,obesity,Alzheimer’s disease,cardiovascular diseases,and cancers.Based on total internal reflection fluorescence microscopy（TIRF）,this thesis developed two single-molecule fluorescence ultrassensitive biosensors for the detection of tumor markers 8-oxoguanine DNA glycosylase and RNA demethylase.The specific research content is as follows:（1）We construct a CRISPR-Cas-based biosensor for rapid and sensitive measurement of 8-oxoguanine DNA glycosylases.This biosensor involves a hairpin probe and integrates quadratic strand displacement amplification（SDA）with a CRISPR/Cas12a effector with the characteristics of rapidity（within 40 min）and isothermal assay.The presence of 8-oxoguanine DNA glycosylase can initiate the quadratic SDA to produce large amounts of activators with the assistance of polynucleotide kinase（PNK）.Subsequently,the activators can bind with cr RNA to activate Cas12a,cleaving signal probes and recovering Cy5 fluorescence,which can be accurately quantified by single-molecule imaging.Notably,the designed hairpin probes can effectively block the hybridization of the generated activators with free hairpin probes,endowing this biosensor with high sensitivity.In addition,the utilization of PNK instead of apurinic/apyrimidinic endonuclease（APE1）greatly simplifies the experimental procedure to only a one-step reaction.The introduction of a single-molecule detection further reduces the sample consumption and improves the sensitivity.This biosensor displays a detection limit of 4.24×10^-9 UμL^-1,and it can accurately quantify cellular human 8-oxoguanine DNA glycosylase（h OGG1）at a single-cell level.Furthermore,this biosensor can be applied for the screening of inhibitors,the analysis of kinetic parameters,and the discrimination of cancer cells from normal cells,with potential applications in molecular diagnostic and point-of-care testing.（2）We developed a label-free single quantum dot（QD）-based nanosensor for the detection of RNA demethylases by combined T7 RNA polymerase（T7）-assisted transcription amplification with the assembly of streptavidin（SA）aptamer-mediated fluorescence resonance energy transfer（FRET）.The proposed nanosensor utilized the hinder effect of N⁶-methyladenosine（m⁶A）in elongation and ligation reactions to distinguish the demethylated RNA and the m⁶A RNA.Therefore,the presence of RNA demethylases can demethylate the m⁶A RNA,and then the demethylated RNA can mediate the elongation and ligation reactions,which can trigger the T7-assisted transcription amplification with the introduction of four kinds of ribonucleotide（ATP,GTP,UTP,and Cy5-CTP）to produce SA aptamers incorporated of Cy5 molecules.The generated SA aptamers can self-assembly to the streptavidin-coated 605 QD to form the 605QD-RNA-Cy5nanostructures,directing FRET between 605QD and Cy5.This nanosensor employed the generated SA aptamers as building materials to construct 605QD-RNA-Cy5 nanostructures,without any involvement of fluorescent or biotin modifications.Moreover,the nanosensor can also be used for the sensitive detection of ALKBH5 demethylase,which makes the nanosensor have universal application potential.This method is extremely sensitive with a detection limit of 4.07f M for FTO,and can accurately detect intracellular FTO at the single-cell level.This nanosensor exhibits ultra-sensitivity,high specificity,and the capability of detection endogenous RNA demethylases at the single-cell level.Moreover,it can be applied for the measurement of enzyme kinetic parameters and the screening inhibitors,and can differentiating the RNA demethylases expression in tissues of healthy persons and breast cancer patients.Such nanosensor has potential applications in clinical diagnosis and of FTO-associated diseases and drug discovery.