Preparation Of Phenylboronic Acid Fluorescent Probe And Its Application In The Analysis Of 5-hydroxymethylcytosine

5-hydroxymethylcytosine(5hmC)is recognized as the sixth base of DNA.It is an important intermediate in active DNA demethylation.It plays important roles in epigenetic reprogramming,regulation of tissue-specific gene expression and degree of cell differentiation.Changes in 5 hmC status have been closely linked to leukemia,autism spectrum disorders,hypertension,aging and various types of cancer so makes itself serve as an important epigenetic mark.Based on the above,high sensitive and selective analysis methods to 5hmC have become a research hotspot.In this study,we developed a simple and sensitive 2-(4-boronophenyl)quinoline-4-carboxylic acid(PBAQA)modified poly glycidyl methacrylate(PBAQA-PGMA)fluorescent probe hope to detect the 5hmC content of genomic DNA in biological samples based on T4 ?-glucosyltransferase-catalyzed glucosylation of 5hmC and the reaction between glucose moiety of glycosyl-modified 5hmC DNA and boric acid group of the PBAQA-PGMA probe.This thesis mainly consists of three parts1.Review:The finding,development,characteristics of distribution in mammals,biological meaning are expounded,the detection methods of 5hmC are conducted a comprehensive review.2.Preparation and performance study of phenylboronic acid functionalized fluorescent probe:Monodisperse,nanoscale PGMA microspheres were prepared by the emulsion polymerization method,the PGMA was modified with ethanediamine and reacted with PBAQA by crosslinking method of EDC/NHS.Finally we got the PBAQA-functionalized fluorescent probe(PBAQA-PGMA).It was characterized by FTIR,XPS and ESEM.The results show that PBAQA was successfully grafted to the surface of PGMA.Scanning electron micrograph revealed that PGMA microspheres were approximately 200 nm in diameter,had a smooth surface,and were monodispersed.When the excitation was 281 nm,the PBAQA-PGMA probe had fluorescence emission at 418 nm.The detection mechanism when the probe recognized and reacted with the G-5hmC-DNA fluorescence enhancement was explored.The test indicated that fluorescence enhancement caused by the G-5hmC-DNA sequence can be explained that binding between the probe and the target,the distance of fluorescent boric acid molecules became larger so the fluorescent signal enhancement.3.Detection of 5hmC by PBAQA-PGMA fluorescence probe:The fluorescence analysis method of 5hmC in biological sampls was established using prepared PBAQA-PGMA fluorescence probe as fluorescence signal source which can recgonize G-5hmC-DNA.Experimetal results show that the probe has good selectivity and recognition to G-5hmC-DNA,the fluorescence-enhanced intensity recorded from the DNA sample was proportional to its 5-hydroxymethylcytosine content and could be quantified by fluorescence spectrophotometry.In the range of 0.5-100nM,the linear correlation of the method is good,the detectionlimit is 0.16nM.The method open up a new way for spectroscopy testing level of 5hmC.The PBAQA-PGMA probe could enrich the content of G-5hmC-DNA from a complex ground substance,it will broaden the linear detection range and improve sensitivity.Contrast with other fluorescence detection methods,this method not only could determine trace amounts of 5 hmC from genomic DNA but also could eliminate the interference of fluorescent dyes and the need for multiple purification.The selectivity and specificity was also improved because of the glucosylation and the specificity recognition of boric acid groups to glucose group in G-5hmC-DNA.