Ratiometric Fluorogenic RNA-Based Sensors for Imaging Live-Cell Dynamics of Small Molecules
Ratiometric Fluorogenic RNA-Based Sensors for Imaging Live-Cell Dynamics of Small Molecules
复制标题
用于小分子活细胞动力学成像的比率荧光 RNA 传感器
DOI：
10.1021/acsabm.9b01237
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发表时间：
2020
影响因子：
4.7
通讯作者：
You, Mingxu
中科院分区：
文献类型：
--
作者：
Wu, Rigumula;Karunanayake Mudiyanselage, Aruni P.K.K.;Ren, Kewei;Sun, Zhining;Tian, Qian;Zhao, Bin;Bagheri, Yousef;Lutati, David;Keshri, Puspam;You, Mingxu
代谢产物和信号分子的细胞动力学成像对于理解各种代谢和信号转导途径至关重要。基于基因编码rna的传感器是实现这一目的的强大工具。然而，使用这些传感器来精确测定目标分析物的细胞内浓度是具有挑战性的。为了解决这个问题，我们最近利用RNA/荧光基团偶联物的正交对开发了比例传感器：西兰花/DFHBI-1T（3,5-二氟-4-羟基苄基-1-三氟乙基-咪唑啉酮）和DNB（二硝基苯胺结合适体）/SR-DN（磺胺-二硝基苯胺）。细胞dnb -西兰花荧光强度比可直接用于定量单细胞水平的靶浓度。不幸的是，由于SR-DN染料的不稳定性，这种比例传感器很难用于监测目标动力学。本文采用稳定的TMR (tetramethylrhodamine)-DN染料替代SR-DN，开发了一种基于Broccoli/DFHBI-1T和DNB/TMR-DN的比例传感器系统，该系统可用于活细胞动态成像。我们相信这些先进的基因编码比例传感器可以广泛用于各种目标分析物的细胞内研究。
Imaging the cellular dynamics of metabolites and signaling molecules is critical for understanding various metabolism and signal transduction pathways. Genetically encoded RNA-based sensors are emerging powerful tools for this purpose. However, it was challenging to use these sensors to precisely determine the intracellular concentrations of target analytes. To solve this problem, we have recently developed ratiometric sensors using an orthogonal pair of RNA/fluorophore conjugates: Broccoli/DFHBI-1T (3,5-difluoro-4-hydroxybenzylidene-1-trifluoroethyl-imidazolinone) and DNB (dinitroaniline-binding aptamer)/SR-DN (sulforhodamine B-dinitroaniline). The cellular DNB-to-Broccoli fluorescence intensity ratio can be directly applied to quantify the target concentrations at the single-cell level. Unfortunately, due to the instability of the SR-DN dye, this ratiometric sensor is difficult to use for monitoring target dynamics. Herein, by replacing SR-DN with a stable TMR (tetramethylrhodamine)-DN dye, we developed a ratiometric sensor system based on Broccoli/DFHBI-1T and DNB/TMR-DN, which can be used for dynamic imaging in living cells. We believe these advanced genetically encoded ratiometric sensors can be widely used for intracellular studies of various target analytes.