University of the Sciences, Department of Chemistry & Biochemistry, 600 S. 43rd St., Philadelphia, PA 19104.
Rowan University, School of Osteopathic Medicine, Department of Cell Biology and Neuroscience, 2 Medical Center Dr., Stratford, NJ 08084.
FEMS Yeast Res. 2018 Dec 1;18(8). doi: 10.1093/femsyr/foy093.
The development of the RNA 'vegetable' aptamers, Spinach and Broccoli, has simplified RNA imaging, especially in live cells. These RNA aptamers interact with a fluorophore (DFHBI or DFHBI-1T) to produce a green fluorescence signal. Although used in mammalian and Escherichia coli cells, the use of these aptamers in yeast has been limited. Here we describe how the Saccharomyces cerevisiae snoRNA, snR30, was tagged with the Spinach or the Broccoli aptamers and observed in live cells. The ability to observe aptamer fluorescence in polyacrylamide gels stained with a fluorophore or with a microplate reader can ease preliminary screening of the aptamers in different RNA scaffolds. In snR30 a tandem repeat of the Broccoli aptamer produced the best signal in vitro. Multiple factors in cell preparation were vital for obtaining a good fluorescence signal. These factors included the clearance of the native unmodified snR30, the amount and length of dye incubation and the rinsing of cells. In cells, the aptamers did not interfere with the structure or essential function of snR30, as the tagged RNA localized to the nucleolus and directed processing of ribosomal RNA in yeast. High-resolution images of the tagged snoRNA were obtained with live cells immobilized by a microcompressor.
RNA“蔬菜”适体（Spinach 和 Broccoli）的发展简化了 RNA 成像，特别是在活细胞中。这些 RNA 适体与荧光团（DFHBI 或 DFHBI-1T）相互作用，产生绿色荧光信号。尽管这些适体已在哺乳动物和大肠杆菌细胞中得到应用，但在酵母中的应用却受到限制。在这里，我们描述了如何在活细胞中标记酿酒酵母 snoRNA（snR30），并观察到 Spinach 或 Broccoli 适体。在聚丙烯酰胺凝胶中用荧光团染色或用微孔板读数器观察适体荧光的能力，可以方便地在不同的 RNA 支架中对适体进行初步筛选。在 snR30 中，Broccoli 适体的串联重复产生了最佳的体外信号。细胞准备过程中的多个因素对于获得良好的荧光信号至关重要。这些因素包括清除天然未修饰的 snR30、染料孵育的量和长度以及细胞的冲洗。在细胞中，适体不会干扰 snR30 的结构或基本功能，因为标记的 RNA 定位于核仁，并指导酵母中核糖体 RNA 的加工。通过微压缩机固定活细胞，可以获得标记 snoRNA 的高分辨率图像。
