title: Fluorescent strategy based on cationic conjugated polymer fluorescence resonance energy transfer for the quantification of 5-(hydroxymethyl)cytosine in genomic DNA.
abstract: DNA methylation is dynamically reprogrammed during early embryonic development in mammals. It can be explained partially by the discovery of 5-(hydroxymethyl)cytosine (5-hmC), 5-formylcytosine (5-fC), and 5-carboxylcytosine (5-caC), which are identified as key players involved in both active and passive demethylation pathways. As one of the ten-eleven translocation oxidation products, 5-hmC was found relatively abundant in neuron cells and embryonic stem cells. Herein we report a new method for 5-hmC quantification in genomic DNA based on CCP-FRET (cationic conjugated polymers act as the energy donor and induce fluorescence resonance energy transfer) assay combined with KRuO4 oxidation. 5-hmC in genomic DNA can be selectively transformed into 5-fC by the oxidation of KRuO4 and then labeled with hydroxylamine-BODIPY (BODIPY = 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) fluorophore through the reaction between 5-fC and hydroxylamine-BODIPY. After the fluorescently labeled DNA was captured by CCP through electrostatic interactions, a significant FRET between CCP and hydroxylamine-BODIPY fluorophore was observed. This CCP-FRET-based assay benefits from light-harvesting, large Stokes shift, and optical signal amplification properties of the CCP. Furthermore, this CCP-FRET-based assay was quite successfully demonstrated for the 5-hmC quantification in three types of cells (mESc, HeLa, HEK 293T), providing a much more convenient choice for 5-hmC quantification in genomic DNA.
authors.affiliation: College of Chemistry and Molecular Sciences, Key Laboratory of Biomedical Polymers of Ministry of Education, Wuhan University , Wuhan, Hubei 430072, People's Republic of China.
authors.forename: Tingting
authors.initials: T
authors.lastname: Hong
authors.forename: Tianlu
authors.initials: T
authors.lastname: Wang
authors.forename: Pu
authors.initials: P
authors.lastname: Guo
authors.forename: Xiwen
authors.initials: X
authors.lastname: Xing
authors.forename: Fei
authors.initials: F
authors.lastname: Ding
authors.forename: Yuqi
authors.initials: Y
authors.lastname: Chen
authors.forename: Jinjun
authors.initials: J
authors.lastname: Wu
authors.forename: Jingwei
authors.initials: J
authors.lastname: Ma
authors.forename: Fan
authors.initials: F
authors.lastname: Wu
authors.forename: Xiang
authors.initials: X
authors.lastname: Zhou
mesh_terms: D044503:5-Methylcytosine / Q000031:analogs & derivatives; D000818:Animals; D002412:Cations / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D003596:Cytosine / Q000031:analogs & derivatives* / Q000032:analysis; D004247:DNA / Q000032:analysis*; D053595:Embryonic Stem Cells / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D010084:Oxidation-Reduction; D011108:Polymers / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D055672:Static Electricity
chemical_list: D002412:Cations; D005456:Fluorescent Dyes; D011108:Polymers; C011865:5-hydroxymethylcytosine; D044503:5-Methylcytosine; D003596:Cytosine; D004247:DNA