Development Of Fluorescent Methods For Sensitive Detection Of Histone Acetylation Writer P300 And Poly (ADP-Ribose) Polymerase-1

Fluorescent technology has the advantages of good specificity,high sensitivity and simple operation,and it has been widely used in many fields such as drug development,disease diagnosis and biogenetic analysis.For example,fluorescent techniques have high sensitivity and high spatial and temporal resolution in measuring dynamics interactions of biomolecules.In addition to the quantitative detection of biomolecules,fluorescent techniques can be used to measure the physiological state of cells or tissues.In this thesis,we constructed two biosensors based on single-molecule fluorescence detection technique and fluorescence spectroscopy detection technique for ultrasensitive detection of histone acetylation writer p300 and poly（ADP-ribose）polymerase-1,respectively.The detailed contents are as follows:1、We demonstrate the simultaneous measurement of histone acetyltransferase（HAT）and histone crotonyltransferase（HCT）activities of the histone acetylation writer p300 by integrating antibody-based fluorescence labeling with single-molecule detection method.The histone acetylation writer p300 has both HAT and HCT activities,which can catalyze histone acetylation/histone crotonylation and are strongly associated with gene expression as well as the development of several diseases.However,the currently developed biosensors focus on the detection of HAT activity,and there is a lack of reliable and efficient methods for the simultaneous detection of HAT and HCT activity.In this work,we used p300 as a model enzyme due to its characteristic dual enzyme activity.We used the N-terminal biotinylated peptide sequence as a substrate for p300 by synthesis.Catalyzes the acetylation/crotonylation of peptide substrates on the corresponding lysine residues when p300 and the acetyl donor acetyl-coenzyme A（Ac Co A）/crotonyl-coenzyme A（Cr Co A）are present.Subsequently,the catalytically produced acetylated/crotonylated peptides can bind to streptavidin-coated magnetic beads（MBs）through streptavidin-biotin interactions.Through antibody-antigen specific interactions,Dy Light 488 and Dy Light 650 labeled anti-histone H3K18 acetylated antibody and anti-histone H3K18 crotonylated antibody,respectively,bind to acetylated/crotonylated peptide magnetic bead complexes to convert the two enzyme activity signals into two distinct fluorescent signals.The dye-labeled peptide substrate is then magnetically separated from the free fluorescent antibody and eluted with deionized water,and the released fluorescent molecules can simply be counted by single-molecule imaging.By measuring the fluorescence signal of Dy Light 488 and Dy Light 650,the HAT and HCT activity of p300 can be quantified simultaneously.The method has good specificity and sensitivity,and the detection limits for HAT are 1.75×10^-4 n M and 6.56×10^-5 n M for HCT.In addition,this method can accurately evaluate the kinetic parameters of HAT and HCT activity of p300 and screen inhibitors.Meanwhile,this method has great potential in clinical diagnosis and drug discovery research.2、We detected the activity of poly（ADP-ribose）polymerase-1（PARP-1）by a fluorescent method based on hyperbranched amplification.PARP-1 catalyzes the formation of poly（ADP-ribose）polymer（PAR polymer）and it widely participates in DNA repair and replication procedures,which is essential for maintaining gene integrity.Accurate and sensitive detection of PARP-1 is critical for clinical diagnosis.However,the current electrostatic attraction-based PAPR-1 methods usually involve laborious procedures,poor sensitivity,and false positive.In this work,taking advantage of unique features of biotinylated nicotinamide adenine dinucleotide（NAD⁺）and terminal deoxynucleotidyl transferase（Td T）,we demonstrate Td T-activated apurinic/apyrimidinic endonuclease 1（APE1）-mediated hyperbranched amplification can be used to detect PARP-1activity by fluorescent method.The specific double-stranded DNA（ds DNA）-activated PARP-1catalyze the transfer of the multiple ADP-ribosyl group of NAD⁺/biotinylated NAD⁺molecules to itself,followed by the formation of ds DNA-PARP-1-PAR polymer bioconjugates containing biotin.Subsequently,the resultant bioconjugates are captured by magnetic beads.The captured ds DNA with dual 3′-OH termini may be amplified by hyperbranched amplification to produce a distinct fluorescence signal.This method exhibits distinct advantages of template-free,label-free,electrostatic interactions-independent,high sensitivity and high accuracy analysis of PARP-1.Moreover,this method convert the PARP-1 enzyme signal to amplifiable DNA signal without the involvement of the laborious preparation and functionalization of nanomaterials,greatly reducing the assay time.This method can sensitively and rapidly measure PARP-1 with a detection limit of4.37×10^-8 U/μL,and it can be applied for detection of cellular PARP-1 at the single-cell level.Importantly,it can screen the PARP-1 inhibitors and accurately discriminate the PARP-1expression level in lung tissues between healthy persons and lung cancer patients,holding great potential in drug discovery and clinical diagnosis.