Zhang Jichuan, Fei Jingyi, Leslie Benjamin J, Han Kyu Young, Kuhlman Thomas E, Ha Taekjip
Department of Physics and Center for the Physics of Living Cells, University of Illinois at Urbana-Champaign, Urbana, IL 61801 USA.
Department of Materials Science and Engineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801 USA.
Sci Rep. 2015 Nov 27;5:17295. doi: 10.1038/srep17295.
Live cell RNA imaging using genetically encoded fluorescent labels is an important tool for monitoring RNA activities. A recently reported RNA aptamer-fluorogen system, the Spinach, in which an RNA aptamer binds and induces the fluorescence of a GFP-like 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI) ligand, can be readily tagged to the RNA of interest. Although the aptamer-fluorogen system is sufficient for imaging highly abundant non-coding RNAs (tRNAs, rRNAs, etc.), it performs poorly for mRNA imaging due to low brightness. In addition, whether the aptamer-fluorogen system may perturb the native RNA characteristics has not been systematically characterized at the levels of RNA transcription, translation and degradation. To increase the brightness of these aptamer-fluorogen systems, we constructed and tested tandem arrays containing multiple Spinach aptamers (8-64 aptamer repeats). Such arrays enhanced the brightness of the tagged mRNA molecules by up to ~17 fold in living cells. Strong laser excitation with pulsed illumination further increased the imaging sensitivity of Spinach array-tagged RNAs. Moreover, transcriptional fusion to the Spinach array did not affect mRNA transcription, translation or degradation, indicating that aptamer arrays might be a generalizable labeling method for high-performance and low-perturbation live cell RNA imaging.
利用基因编码荧光标记进行活细胞RNA成像，是监测RNA活性的一项重要工具。最近报道的一种RNA适体-荧光原系统——菠菜（Spinach）系统，其中的RNA适体能够结合并诱导一种类绿色荧光蛋白的3,5-二氟-4-羟基苯亚甲基咪唑啉酮（DFHBI）配体发出荧光，可轻松标记到感兴趣的RNA上。尽管适体-荧光原系统足以对高丰度非编码RNA（tRNA、rRNA等）进行成像，但由于亮度较低，其在mRNA成像方面表现不佳。此外，适体-荧光原系统是否会干扰天然RNA的特性，尚未在RNA转录、翻译和降解水平上进行系统表征。为了提高这些适体-荧光原系统的亮度，我们构建并测试了包含多个菠菜适体（8 - 64个适体重复序列）的串联阵列。这种阵列在活细胞中将标记的mRNA分子亮度提高了约17倍。用脉冲照明进行强激光激发进一步提高了菠菜阵列标记RNA的成像灵敏度。此外，与菠菜阵列的转录融合并不影响mRNA的转录、翻译或降解，这表明适体阵列可能是一种适用于高性能、低干扰活细胞RNA成像的通用标记方法。
