Department of Chemistry, University of Massachusetts, Amherst, MA, USA.
Methods Mol Biol. 2025;2875:21-36. doi: 10.1007/978-1-0716-4248-1_3.
The advancement of nucleic acid nanotechnology has resulted in broad applications of DNA- and RNA-based molecular sensors for bioanalysis. Catalytic hairpin assembly is such a type of programmable and enzyme-free nucleic acid circuit that has been popularly used in developing biosensors. Genetically encodable fluorogenic RNA-based devices have recently gained a lot of attentions as a powerful tool for intracellular imaging. Combining these two techniques together, we have developed a genetically encodable RNA-based catalytic hairpin assembly circuit for the highly sensitive detection of low-abundance RNAs in living cells. In this system, the binding of one target RNA can catalytically trigger the generation of tens-to-hundreds of fluorogenic RNA reporters and produce a significantly enhanced fluorescence signal. Here, we will introduce the detailed design, optimization, and imaging protocol of this new type of powerful imaging tools.
核酸纳米技术的进步使得基于 DNA 和 RNA 的分子传感器在生物分析中有了广泛的应用。催化发夹组装就是这样一种可编程且无酶的核酸电路，已广泛应用于生物传感器的开发中。基于遗传编码的荧光 RNA 器件最近作为一种强大的细胞内成像工具引起了广泛关注。将这两种技术结合在一起，我们开发了一种基于遗传编码的 RNA 催化发夹组装电路，用于在活细胞中高灵敏度地检测低丰度的 RNA。在这个系统中，一个靶 RNA 的结合可以催化引发数十到数百个荧光 RNA 报告分子的生成，并产生显著增强的荧光信号。在这里，我们将介绍这种新型强大成像工具的详细设计、优化和成像方案。
