title: Spectroscopic quantification of 5-hydroxymethylcytosine in genomic DNA using boric acid-functionalized nano-microsphere fluorescent probes.
abstract: 5-hydroxymethylcytosine (5hmC) is the sixth base of DNA. It is involved in active DNA demethylation and can be a marker of diseases such as cancer. In this study, we developed a simple and sensitive 2-(4-boronophenyl)quinoline-4-carboxylic acid modified poly (glycidyl methacrylate (PBAQA-PGMA) fluorescent probe to detect the 5hmC content of genomic DNA based on T4 β-glucosyltransferase-catalyzed glucosylation of 5hmC. The fluorescence-enhanced intensity recorded from the DNA sample was proportional to its 5-hydroxymethylcytosine content and could be quantified by fluorescence spectrophotometry. The developed probe showed good detection sensitivity and selectivity and a good linear relationship between the fluorescence intensity and the concentration of 5 hmC within a 0-100nM range. Compared 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 purification. It also could avoid multiple labeling. Because the PBAQA-PGMA probe could enrich the content of glycosyl-5-hydroxymethyl-2-deoxycytidine from a complex ground substance, it will broaden the linear detection range and improve sensitivity. The limit of detection was calculated to be 0.167nM after enrichment. Furthermore, the method was successfully used to detect 5-hydroxymethylcytosine from mouse tissues.
authors.affiliation: Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry & Chemical Engineering, Shaanxi Normal University, Xi'an 710062, China.
authors.forename: Hua-Yan
authors.initials: HY
authors.lastname: Chen
authors.affiliation: Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry & Chemical Engineering, Shaanxi Normal University, Xi'an 710062, China.
authors.forename: Jing-Ru
authors.initials: JR
authors.lastname: Wei
authors.affiliation: Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry & Chemical Engineering, Shaanxi Normal University, Xi'an 710062, China.
authors.forename: Jiong-Xiu
authors.initials: JX
authors.lastname: Pan
authors.affiliation: Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry & Chemical Engineering, Shaanxi Normal University, Xi'an 710062, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry & Chemical Engineering, Shaanxi Normal University, Xi'an 710062, China.
authors.forename: Fu-Quan
authors.initials: FQ
authors.lastname: Dang
authors.affiliation: Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry & Chemical Engineering, Shaanxi Normal University, Xi'an 710062, China.
authors.forename: Zhi-Qi
authors.initials: ZQ
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry & Chemical Engineering, Shaanxi Normal University, Xi'an 710062, China. Electronic address: zhangjing8902@snnu.edu.cn.
authors.forename: Jing
authors.initials: J
authors.lastname: Zhang
mesh_terms: D044503:5-Methylcytosine / Q000031:analogs & derivatives* / Q000032:analysis; D000818:Animals; D001888:Boric Acids / Q000737:chemistry*; D001923:Brain Chemistry; D004247:DNA / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry*; D008099:Liver / Q000737:chemistry; D051379:Mice; D009206:Myocardium / Q000737:chemistry; D011109:Polymethacrylic Acids / Q000737:chemistry*; D011804:Quinolines / Q000737:chemistry; D013050:Spectrometry, Fluorescence / Q000379:methods*
chemical_list: D001888:Boric Acids; D005456:Fluorescent Dyes; D011109:Polymethacrylic Acids; D011804:Quinolines; C011865:5-hydroxymethylcytosine; C042535:polyglycidyl methacrylate; C059924:quinoline-4-carboxylic acid; D044503:5-Methylcytosine; D004247:DNA; C032688:boric acid
keywords: 5-hydroxymethylcytosine; Enrichment; Fluorescent probe; Genomic DNA; Glucosylation