School of Biological Sciences and Institute of Biological and Biomedical Sciences, University of Portsmouth, Portsmouth PO1 2DY, United Kingdom.
School of Biological Sciences and Institute of Biological and Biomedical Sciences, University of Portsmouth, Portsmouth PO1 2DY, United Kingdom.
Methods. 2019 Sep 1;167:39-53. doi: 10.1016/j.ymeth.2019.04.021. Epub 2019 May 2.
The discovery and engineering of more and more functions of RNA has highlighted the utility of RNA-targeting small molecules. Recently, several fluorogen-binding RNA aptamers have been developed that have been applied to live cell imaging of RNA and metabolites as RNA tags or biosensors, respectively. Although the design and application of these fluorogen-binding RNA aptamer-based devices is straightforward in theory, in practice, careful optimisation is required. For this reason, high throughput in vitro screening techniques, capable of quantifying fluorogen-RNA aptamer interactions, would be beneficial. We recently developed a method for generating functional-RNA arrays and demonstrated that they could be used to detect fluorogen-RNA aptamer interactions. Specifically, we were able to visualise the interaction between malachite green and the malachite green-binding aptamer. Here we expand this study to demonstrate that functional-RNA arrays can be used to quantify fluorogen-aptamer interactions. As proof-of-concept, we provide detailed protocols for the production of malachite green-binding RNA aptamer and DFHBI-binding Spinach RNA aptamer arrays. Furthermore, we discuss the potential utility of the technology to fluorogen-binding RNA aptamers, including application as a molecular biosensor platform. We anticipate that functional-RNA array technology will be beneficial for a wide variety of biological disciplines.
越来越多的 RNA 功能的发现和工程化，突出了 RNA 靶向小分子的实用性。最近，已经开发了几种荧光染料结合 RNA 适体，它们分别被应用于 RNA 和代谢物的活细胞成像作为 RNA 标记物或生物传感器。虽然这些基于荧光染料结合 RNA 适体的器件的设计和应用在理论上是简单直接的，但实际上需要仔细优化。因此，高通量的体外筛选技术，能够定量荧光染料-RNA 适体相互作用，将是有益的。我们最近开发了一种生成功能 RNA 阵列的方法，并证明它们可用于检测荧光染料-RNA 适体相互作用。具体来说，我们能够可视化孔雀石绿与孔雀石绿结合适体之间的相互作用。在这里，我们扩展了这项研究，证明功能 RNA 阵列可用于定量荧光染料-适体相互作用。作为概念验证，我们提供了详细的孔雀石绿结合 RNA 适体和 DFHBI 结合 Spinach RNA 适体阵列的生产方案。此外，我们讨论了该技术在荧光染料结合 RNA 适体中的潜在应用，包括作为分子生物传感器平台的应用。我们预计功能 RNA 阵列技术将有益于广泛的生物学领域。
