Imaging Intracellular S-Adenosyl Methionine Dynamics in Live Mammalian Cells with a Genetically Encoded Red Fluorescent RNA-Based Sensor.
Imaging Intracellular S-Adenosyl Methionine Dynamics in Live Mammalian Cells with a Genetically Encoded Red Fluorescent RNA-Based Sensor.
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DOI：
10.1021/jacs.0c02931
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发表时间：
2020-08-19
影响因子：
15
通讯作者：
Jaffrey SR
中科院分区：
文献类型：
--
作者：
Li X;Mo L;Litke JL;Dey SK;Suter SR;Jaffrey SR
为了了解细胞内代谢产物在细胞过程中的作用，重要的是要测量活细胞中小分子的动力学和通量。虽然传统的由荧光蛋白组成的代谢物传感器已被用于检测某些代谢物，但一种新兴的方法是使用由RNA组成的遗传编码传感器。由于能够快速生成代谢物结合RNA适体，基于RNA的传感器具有比基于蛋白质的传感器更容易设计的潜力。已经开发了许多策略来将绿色荧光菠菜或西兰花荧光RNA适体转化为代谢物调节的传感器。然而，由于细胞中的低水平红色背景荧光，红色荧光是特别期望的。然而，当用其同源荧光团成像时，花椰菜适体的红色荧光变体红花椰菜在细胞中不显示红色荧光。目前还不清楚为什么红花椰菜在体外有荧光，而在活的哺乳动物细胞中没有。在这里，我们开发了一种新的荧光团，OBI（3，5-二氟-4-羟基苯亚甲基-咪唑啉酮-2-肟-1-苯并咪唑），它与红花椰菜结合具有高亲和力，使红花椰菜抵抗热解折叠。我们表明，OBI使红花椰菜很容易在活的哺乳动物细胞中检测到。此外，我们表明，红花椰菜可以融合到一个S-腺苷甲硫氨酸结合适体，以产生一个红色的荧光RNA为基础的传感器，使SAM在活的哺乳动物细胞成像。这些结果揭示了在哺乳动物细胞中起作用的红色荧光荧光适体，并且其可以容易地开发成基于红色荧光RNA的传感器。
In order to understand the role of intracellular metabolites in cellular processes, it is important to measure the dynamics and fluxes of small molecules in living cells. Although conventional metabolite sensors composed of fluorescent proteins have been made to detect some metabolites, an emerging approach is to use genetically encoded sensors composed of RNA. Because of the ability to rapidly generate metabolite-binding RNA aptamers, RNA-based sensors have the potential to be designed more readily than protein-based sensors. Numerous strategies have been developed to convert the green-fluorescent Spinach or Broccoli fluorogenic RNA aptamers into metabolite-regulated sensors. Nevertheless, red fluorescence is particularly desirable due to low level of red background fluorescence in cells. However, the red fluorescent variant of the Broccoli aptamer, Red Broccoli, does not exhibit red fluorescence in cells when imaged with its cognate fluorophore. It is not known why Red Broccoli is fluorescent in vitro but not in live mammalian cells. Here we develop a new fluorophore, OBI (3,5-difluoro-4-hydroxybenzylidene-imidazolinone-2-oxime-1-benzoimidazole), which binds Red Broccoli with high affinity and makes Red Broccoli resistant to thermal unfolding. We show that OBI enables Red Broccoli to be readily detected in live mammalian cells. Furthermore, we show that Red Broccoli can be fused to a S-adenosyl methionine-binding aptamer to generate a red fluorescent RNA-based sensor that enables imaging of SAM in live mammalian cells. These results reveal a red fluorescent fluorogenic aptamer that functions in mammalian cells and which can be readily developed into red fluorescent RNA-based sensors.
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DOI：
10.1126/science.1207339
发表时间：
2011-07-29
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Paige JS;Wu KY;Jaffrey SR
通讯作者：
Jaffrey SR
DOI：
10.1126/science.aat4422
发表时间：
2018-06-29
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Patriarchi T;Cho JR;Merten K;Howe MW;Marley A;Xiong WH;Folk RW;Broussard GJ;Liang R;Jang MJ;Zhong H;Dombeck D;von Zastrow M;Nimmerjahn A;Gradinaru V;Williams JT;Tian L
通讯作者：
Tian L
基因编码荧光传感器揭示 NADPH 代谢的动态调节
影响因子：
48
作者：
Tao R;Zhao Y;Chu H;Wang A;Zhu J;Chen X;Zou Y;Shi M;Liu R;Su N;Du J;Zhou HM;Zhu L;Qian X;Liu H;Loscalzo J;Yang Y
通讯作者：
Yang Y
影响因子：
48
作者：
Cameron, William D.;Bui, Cindy V.;Rocheleau, Jonathan V.
通讯作者：
Rocheleau, Jonathan V.
影响因子：
16.6
作者：
Lobas, Mark A.;Tao, Rongkun;Khakh, Baljit S.
通讯作者：
Khakh, Baljit S.