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Identification and Quantification of 5-Methylcytosine and 5-Hydroxymethylcytosine on Random DNA Sequences by a Nanoconfined Electrochemiluminescence Platform
Mao-Hua Gao, Mei-Chen Pan, Pu Zhang, Wen-Bin Liang, Xia Zhong*, and Ying Zhuo*
Cite this: Anal. Chem. 2023, 95, 25, 9598–9604
Publication Date:June 13, 2023
https://doi.org/10.1021/acs.analchem.3c01252


Abstract


5-Methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are two of the most abundant epigenetic marks in mammalian genomes, and it has been proven that these dual epigenetic marks give a more accurate prediction of recurrence and survival in cancer than the individual mark. However, due to the similar structure and low expression of 5mC and 5hmC, it is challenging to distinguish and quantify the two methylation modifications. Herein, we employed the ten-eleven translocation family dioxygenases (TET) to convert 5mC to 5hmC via a specific labeling process, which realized the identification of the two marks based on a nanoconfined electrochemiluminescence (ECL) platform combined with the amplification strategy of a recombinase polymerase amplification (RPA)-assisted CRISPR/Cas13a system. Benefiting from the TET-mediated conversion strategy, a highly consistent labeling pathway was developed for identifying dual epigenetic marks on random sequence, which reduced the system error effectively. The ECL platform was established via preparing a carbonized polymer dot embedded SiO2 nanonetwork (CPDs@SiO2), which exhibited higher ECL efficiencies and more stable ECL performance compared to those of the scattered emitters due to the nanoconfinement-enhanced ECL effect. The proposed bioanalysis strategy could be employed for the identification and quantification of 5mC and 5hmC in the range from 100 aM to 100 pM, respectively, which provides a promising tool for early diagnosis of diseases associated with abnormal methylation.

5-甲基胞嘧啶（5mC）和5-羥甲基胞嘧啶是哺乳動(dòng)物基因組中最豐富的兩種表觀遺傳標(biāo)記，并且已經(jīng)證明，這些雙重表觀遺傳標(biāo)志比個(gè)體標(biāo)記更準(zhǔn)確地預(yù)測(cè)癌癥的復(fù)發(fā)和存活。然而，由于5mC和5hmC的結(jié)構(gòu)相似且低表達(dá)，區(qū)分和量化這兩種甲基化修飾具有挑戰(zhàn)性。在此，我們使用了十個(gè)十一位易位家族的雙加氧酶（TET），通過(guò)特定的標(biāo)記過(guò)程將5mC轉(zhuǎn)化為5hmC，這實(shí)現(xiàn)了基于納米限制電化學(xué)發(fā)光（ECL）平臺(tái)的兩個(gè)標(biāo)記的識(shí)別，并結(jié)合了重組酶聚合酶擴(kuò)增（RPA）輔助的CRISPR/Cas13a系統(tǒng)的擴(kuò)增策略。得益于TET介導(dǎo)的轉(zhuǎn)化策略，開(kāi)發(fā)了一種高度一致的標(biāo)記途徑來(lái)識(shí)別隨機(jī)序列上的雙重表觀遺傳標(biāo)記，有效地減少了系統(tǒng)誤差。ECL平臺(tái)是通過(guò)制備碳化聚合物點(diǎn)嵌入SiO2納米網(wǎng)絡(luò)而建立的(CPDs@SiO2)，由于納米約束增強(qiáng)的ECL效應(yīng)，與散射發(fā)射體相比，其表現(xiàn)出更高的ECL效率和更穩(wěn)定的ECL性能。所提出的生物分析策略可用于分別鑒定和定量100aM至100pM范圍內(nèi)的5mC和5hmC，這為早期診斷與異常甲基化相關(guān)的疾病提供了一個(gè)有前途的工具。

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