Fluorescent Strategy Based on Cationic Conjugated Polymer Fluorescence Resonance Energy Transfer for the Quantification of 5-(Hydroxymethyl)cytosine in Genomic DNA

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• 作者：Tingting Hong ; Tianlu Wang ; Pu Guo ; Xiwen Xing ; Fei Ding ; Yuqi Chen ; Jinjun Wu ; Jingwei Ma ; Fan Wu ; Xiang Zhou
• 刊名：Analytical Chemistry
• 出版年：2013
• 出版时间：November 19, 2013
• 年：2013
• 卷：85
• 期：22
• 页码：10797-10802
• 全文大小：327K
• 年卷期：v.85,no.22(November 19, 2013)
• ISSN：1520-6882

文摘

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鈥揺leven 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.