Fluorescence Detection Of Protein Kinase And N~6-Methyladenine

The protein and nucleic acids is the carrier of epigenetic information,which play an imporment role in the field of life science.Fluorescence technology is an advanced analytical method,which is widely employed in life science research,such as drug research and clinic diagnostics.The steady-state fluorescence detection and the single molecule fluorescence detection are important methods for fluorescence analysis of proteins and nucleic acids.As the most common fluorescence analysis method,steady-state fluorescence detection can realize the qualitative and quantitative analysis of biomolecules through fluorescence spectrum and fluorescence intensity.Compared with traditional integrated measurement methods,single molecule fluorescence detection has the advantages of high sensitivity,good selectivity,fast and low sample consumption,and it can be used as an ideal analytical method to quickly and simply quantify low abundance biomolecules.In this thesis,we used steady-state fluorescence spectrometry measurement and single-molecule fluorescence detection（SMD）to detect protein kinase and N⁶-methyladenosine（m⁶A）,respectively.This thesis mainly includes the following contents:（1）Protein kinases play important roles in regulating various cellular processes and may function as potential diagnostic and therapeutic targets for various diseases including cancers.Herein,we construct a phos-tag-directed self-assembled fluorescent magnetobiosensor to simultaneously detect multiple protein kinases with good selectivity and high sensitivity.In the presence of protein kinases（PKA and Akt1）,their substrate peptides（a FITC-labeled substrate peptide and a Cy5-labeled substrate peptide）are phosphorylated,and are then specifically recognized and captured by a biotinylated phos-tag to generate biotinylated substrate peptides for the assembly of magnetic bead（MB）-peptides-FITC/Cy5 nanostructures.After magnetic separation,the phosphorylated substrate peptides are disassembled from the MB-peptides-FITC/Cy5 nanostructures using deionized water at 80℃,releasing FITC and Cy5 molecules.The released FITC and Cy5 molecules are detected by steady-state fluorescence measurements,with FITC indicating PKA and Cy5 indicating Akt1.This magnetobiosensor only involves one phos-tag without the requirement of radiolabeling,antibody screening,carboxypeptidase Y（CPY）cleavage,and cumbersome chemical/enzyme reactions.The introduction of magnetic separation can effectively eliminate the interference from complex real samples,generating an extremely low background signal.Moreover,this magnetobiosensor can accurately measure cellular protein kinase activities and screen inhibitors,with great potential for kinase-related biomedical research and therapeutic applications.（2）m⁶A is the most abundant chemical modification in eukaryotic m RNA,and it is known to participate in a variety of physiological processes,including cancer progression and viral infection.The development of m⁶A detection technology is of great importance for studying the biological functions of m⁶A.Herein,we develop a single-quantum dot（QD）nanosensor to accurately detect m⁶A based on copper-free and enzyme-free cycling click chemistry-mediated ligation detection reaction（LDR）amplification.We design four DNA probes including a-d,azide（N₃）-labeled probe a,DBCO-modified probe b,biotin and N₃-labeled probe c,DBCO and Cy5-labeled probe d.When m⁶A is absent,the RNA sequence is cleaved by the Maz F enzyme,and when m⁶A is present,the RNA sequence is retained.In the presence of target m⁶A-RNA,probes a and probe b are brought into close proximity due to the hybridization of target with probes a and probe b,followed by the formation of sandwich-type hybrid.The resultant sandwich-type hybrid leads to the initiation of click chemical reaction between the DBCO and N₃ groups,followed by the formation of probes a-b ligation product.Then through the denaturation reaction,the released m⁶A-RNA can act as a template to hybridize with more DNA probes a and DNA probes b,initiating the first LDR through the thermal cycle of 95℃and 25℃.Meanwhile,the resultant probes a-b ligation product can be used as the template to hybridize with DNA probe c and DNA probe d.The DBCO and N₃ groups in the DNA probe d and DNA probe c,respectively,can form the triazol linkage after the copper-free click chemical reaction.The denaturation at 95℃induces the probe a-b ligation products can be used as the template to hybridize with more DNA probes c and DNA probes d,initiating the tandem LDR amplification through the thermal cycle of 95℃and 25℃,generating abundant probe c-d ligation products with biotin modified at the 5’end and Cy5 modified near the 3’end.The resultant probe c-d ligation product binds to QD to generate FRET signal.This assay does not involve either enzymatic reverse transcription,copper catalyst,or ligase enzyme,and it exhibits excellent selectivity and high sensitivity(a detection limit of 1.42×10^-15 M).Moreover,this nanosensor can even discriminate 0.01%m⁶A-RNA in the mixture of m⁶A-RNA and A-RNA,and it can accurately quantify m⁶A in caner cells.