State Key Laboratory of Analytical Chemistry for Life Science and Collaborative Innovation Center of Chemistry for Life Sciences, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023, China.
Institute for Materials Science and Engineering, School of Materials Sciences and Engineering, Suzhou University of Science and Technology, Suzhou 215011, China.
ACS Sens. 2021 Jun 25;6(6):2339-2347. doi: 10.1021/acssensors.1c00453. Epub 2021 May 24.
As the cellular roles of RNA abundance continue to increase, there is an urgent need for the corresponding tools to elucidate native RNA functions and dynamics, especially those of short, low-abundance RNAs in live cells. Fluorescent RNA aptamers provide a useful strategy to create the RNA tag and biosensor devices. Corn, which binds with 3,5-difluoro-4-hydroxybenzylidene-imidazolinone-2-oxime (DFHO), is a good candidate for the RNA tag because of its enhanced photostability and red-shifted spectrum. Herein, we report for the first time the utilization of Corn as a split aptamer system, combined with RNA-initiated fluorescence complementation (RIFC), for monitoring RNA self-assembly and sensing microRNA. In this platform, the 28-nt Corn was divided into two nonfunctional halves (named probe I and probe II), and an additional target RNA recognition and stem part was introduced in each probe. The target RNA can trigger the self-assembly reconstitution of the Corn's G-quadruplex scaffold for DFHO binding and turn-on fluorescence. These probes can be transfected stably into mammalian cells and deliver the light-up fluorescent response to microRNA-21 (miR-21). Significantly, the probes have good photostability, with minimal fluorescence loss after continuous irradiation, and can be used for imaging of miR-21 in living mammalian cells. The proposed method is universal and could be applied to the sensing of other tumor-associated RNAs, including messenger RNA and noncoding RNA, as well as for monitoring RNA/RNA interactions. The Corn-based splitting aptamers show promising potential in the real-time visualization and mechanistic analysis of nucleic acids.
随着 RNA 丰度的细胞功能不断增加，迫切需要相应的工具来阐明天然 RNA 的功能和动态，特别是在活细胞中那些短的、低丰度的 RNA。荧光 RNA 适体为创建 RNA 标记和生物传感器设备提供了一种有用的策略。玉米结合 3,5-二氟-4-羟基苯亚甲基-咪唑啉-2-恶啉（DFHO），是 RNA 标记的良好候选物，因为它具有增强的光稳定性和红移光谱。在此，我们首次报道了将 Corn 用作分裂适体系统，结合 RNA 引发的荧光互补（RIFC），用于监测 RNA 自组装和感应 microRNA。在这个平台中，28 个核苷酸的 Corn 被分成两个非功能的部分（分别命名为探针 I 和探针 II），并且在每个探针中引入了一个额外的目标 RNA 识别和茎部分。目标 RNA 可以触发 Corn 的 G-四链体支架的自组装重组成分，用于 DFHO 结合和开启荧光。这些探针可以稳定转染到哺乳动物细胞中，并将点亮的荧光响应传递给 microRNA-21（miR-21）。值得注意的是，这些探针具有良好的光稳定性，在连续照射后荧光损失最小，可用于活哺乳动物细胞中 miR-21 的成像。该方法具有通用性，可应用于其他与肿瘤相关的 RNA 的传感，包括信使 RNA 和非编码 RNA，以及用于监测 RNA/RNA 相互作用。基于 Corn 的分裂适体在核酸的实时可视化和机制分析方面显示出有前景的潜力。
