Institute of Bioengineering and Bioimaging, A*STAR, 31 Biopolis Way, The Nanos #07-01, Singapore, 138669, Singapore.
Chiba Institute of Technology (CIT), Tsudanuma 2-17-1, Narashino, Chiba 275-0016, Japan.
Chemistry. 2022 Mar 16;28(16):e202104396. doi: 10.1002/chem.202104396. Epub 2022 Feb 24.
Light-emitting systems using an RNA aptamer-dye pair, such as Spinach RNA, are an attractive method for imaging and tracing RNA expression in vitro and in vivo. We present an alternative Spinach method by genetic alphabet expansion using an unnatural base pair system, in which a dye-conjugated unnatural base substrate is site-specifically incorporated at a specific position in Spinach RNA by transcription involving the third base pair. The incorporation position was predicted by molecular dynamics simulations. This dye-conjugated Spinach RNA increased the thermal stability of the fluorescence, the robustness against ion sensitivity, and the resistance against photobleaching. Furthermore, we applied our method to Baby Spinach, a shorter version of Spinach, for dye conjugation toward the visible detection of transcripts. This is the first demonstration of an alternative RNA imaging method for a detection system using genetic alphabet expansion.
使用 RNA 适体-染料对的发光系统，如 Spinach RNA，是体外和体内成像和追踪 RNA 表达的一种有吸引力的方法。我们通过遗传字母扩展展示了一种替代的 Spinach 方法，该方法使用非天然碱基对系统，其中通过涉及第三碱基对的转录，将染料缀合的非天然碱基底物特异性地掺入 Spinach RNA 的特定位置。掺入位置通过分子动力学模拟预测。这种与染料共轭的 Spinach RNA 增加了荧光的热稳定性、对离子敏感性的稳健性和对光漂白的抵抗力。此外，我们将我们的方法应用于 Baby Spinach，即 Spinach 的较短版本，用于染料缀合，以实现对转录物的可见光检测。这是使用遗传字母扩展的检测系统的替代 RNA 成像方法的首次演示。
