基因編碼FRET熒光傳感器，被設(shè)計(jì)用于在體外和活細(xì)胞中檢測(cè)不對(duì)稱二甲基化水平

2023年2月，中國(guó)科學(xué)技術(shù)大學(xué)基礎(chǔ)醫(yī)學(xué)院生命科學(xué)與醫(yī)學(xué)部中國(guó)科學(xué)院先天免疫與慢性病重點(diǎn)實(shí)驗(yàn)室合肥微尺度物理科學(xué)國(guó)家實(shí)驗(yàn)室;中國(guó)科學(xué)技術(shù)大學(xué)**附屬醫(yī)院(Hefei National Laboratory for Physical Sciences at Microscale, the CAS Key Laboratory of Innate Immunity and Chronic Disease, School of Basic Medical Sciences, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei 230027, China；The First Afliated Hospital of University of Science and Technology of China, Hefei 230001, China) Xuan Sun老師研究團(tuán)隊(duì)在《Analytical and Bioanalytical Chemistry》上發(fā)表論文：

“A gene-encoded FRET fluorescent sensor designed for detecting asymmetric dimethylation levels in vitro and in living cells”

“基因編碼FRET熒光傳感器，被設(shè)計(jì)用于在體外和活細(xì)胞中檢測(cè)不對(duì)稱二甲基化水平”

Abstract：

Arginine methylation is involved in many important biological processes. PRMT1 is a major arginine methyltransferase in mammalian cells and is highly conserved in eukaryotes. It catalyzes the methylation of various of substrates, including histones, and PRMT1 has been reported to be overexpressed in many cancers, indicating that it is a potential therapeutic target. No tool for efficient methylation level detection in living cells has been available to date. In this work, we designed and constructed a gene-encoded fluorescence resonance energy transfer (FRET) fluorescent sensor for detecting dimethylation levels in living cells and evaluated its functional efficiency both in vitro and in living cells. Both site-directed mutagenesis and PRMT1 inhibition experiments verified that the fluorescent sensor responded to changes in PRMT1 activity and to different PRMT1-induced methylation levels in vitro. Finally, we verified that this optimized methyl sensor responded sensitively to changes in methylation levels in living cells by overexpressing and inhibiting PRMT1, which makes it a useful tool for real-time imaging of arginine methylation. As a new tool for detecting arginine dimethylation levels in living cells, the designed FRET sensor is very important for posttranslational studies and may show a wide range of applications.

摘要：

精氨酸甲基化參與了許多重要的生物過(guò)程。PRMT1是哺乳動(dòng)物細(xì)胞中主要的精氨酸甲基轉(zhuǎn)移酶，在真核生物中高度保守。它催化包括組蛋白在內(nèi)的多種底物的甲基化，據(jù)報(bào)道，PRMT1在許多癌癥中過(guò)表達(dá)，這表明它是一個(gè)潛在的治療靶點(diǎn)。迄今為止，還沒(méi)有有效的活細(xì)胞甲基化水平檢測(cè)工具。

在這項(xiàng)工作中，科研人員設(shè)計(jì)并構(gòu)建了一個(gè)基因編碼熒光共振能量轉(zhuǎn)移(FRET)熒光傳感器，用于檢測(cè)活細(xì)胞中的二甲基化水平，并評(píng)估了其在體外和活細(xì)胞中的功能效率。位點(diǎn)定向誘變和PRMT1抑制實(shí)驗(yàn)都證實(shí)了熒光傳感器對(duì)PRMT1活性的變化和不同PRMT1誘導(dǎo)的體外甲基化水平有響應(yīng)。最后，科研人員驗(yàn)證了這種優(yōu)化的甲基傳感器通過(guò)過(guò)表達(dá)和抑制PRMT1對(duì)活細(xì)胞中甲基化水平的變化做出敏感的反應(yīng)，這使其成為精氨酸甲基化實(shí)時(shí)成像的有用工具。作為一種檢測(cè)活細(xì)胞中精氨酸二甲基化水平的新工具，所設(shè)計(jì)的FRET傳感器在翻譯后研究中具有重要意義，具有廣泛的應(yīng)用前景。

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