Filonov Grigory S, Kam Christina W, Song Wenjiao, Jaffrey Samie R
Department of Pharmacology, Weill Medical College, Cornell University, New York, NY 10065, USA.
Department of Pharmacology, Weill Medical College, Cornell University, New York, NY 10065, USA.
Chem Biol. 2015 May 21;22(5):649-60. doi: 10.1016/j.chembiol.2015.04.018.
RNA aptamers can be expressed in cells to influence and image cellular processes. Aptamer folding is maintained by inserting the aptamers into highly structured RNA scaffolds. Here, we show that commonly used RNA scaffolds exhibit unexpected instability and cleavage in bacterial and mammalian cells. Using an in-gel staining approach for rapid and simple detection of Spinach- or Broccoli-tagged RNAs in cells, we monitored the processing of RNAs tagged with scaffolded aptamers, revealing endonucleolytic cleavage, RNA instability, and poor expression. We reengineered a natural three-way junction structure to generate an alternative scaffold that enables stable aptamer expression in cells. This scaffold was used to create cassettes containing up to four Broccoli units, markedly enhancing the brightness of mammalian cells expressing cassette-tagged RNAs. These experiments describe methods for screening RNA cleavage events in cells and identify cell-compatible scaffolds that enable efficient tagging of RNAs with aptamers for cellular expression.
RNA适配体可在细胞中表达，以影响细胞过程并对其进行成像。通过将适配体插入高度结构化的RNA支架中可维持适配体折叠。在此，我们表明常用的RNA支架在细菌和哺乳动物细胞中表现出意想不到的不稳定性和切割现象。使用凝胶内染色方法快速简单地检测细胞中带有菠菜或西兰花标签的RNA，我们监测了带有支架适配体标签的RNA的加工过程，揭示了核酸内切酶切割、RNA不稳定性和低表达情况。我们对天然的三向接头结构进行了重新设计，以生成一种替代支架，该支架能够在细胞中实现稳定的适配体表达。这种支架被用于创建包含多达四个西兰花单元的盒式结构，显著增强了表达盒式标签RNA的哺乳动物细胞的亮度。这些实验描述了筛选细胞中RNA切割事件的方法，并鉴定出与细胞兼容的支架，这些支架能够用适配体对RNA进行有效标记以用于细胞表达。
