Guzmán-Zapata Daniel, Domínguez-Anaya Yael, Macedo-Osorio Karla S, Tovar-Aguilar Andrea, Castrejón-Flores José L, Durán-Figueroa Noé V, Badillo-Corona Jesús A
Instituto Politécnico Nacional, Unidad Profesional Interdisciplinaria de Biotecnología, Av. Acueducto S/N., Col. Barrio La Laguna Ticomán, 07340 México City, Mexico.
Instituto Politécnico Nacional, Unidad Profesional Interdisciplinaria de Biotecnología, Av. Acueducto S/N., Col. Barrio La Laguna Ticomán, 07340 México City, Mexico.
J Biotechnol. 2017 Jun 10;251:186-188. doi: 10.1016/j.jbiotec.2017.03.028. Epub 2017 Mar 28.
Light-up aptamers are practical tools to image RNA localization in vivo. A now classical light-up aptamer system is the combination of the 3,5-difluoro-4-hydroxybenzylidene (DFHBI) fluorogen and the RNA aptamer Spinach, which has been successfully used in bacterial and mammalian cells. However, light-up aptamers have not been used in algae. Here, we show that a simple vector, carrying Spinach, transcriptionally fused to the aphA-6 gene, can be effectively used to generate a functional light-up aptamer in the chloroplast of Chlamydomonas reinhardtii. After incubation with DFHBI, lines expressing the aphA-6/Spinach mRNA were observed with laser confocal microscopy to evaluate the functionality of the light-up aptamer in the chloroplast of C. reinhardtii. Clear and strong fluorescence was localized to the chloroplast, in the form of discrete spots. There was no background fluorescence in the strain lacking Spinach. Light-up aptamers could be further engineered to image RNA or to develop genetically encoded biosensors in algae.
点亮型适体是用于体内RNA定位成像的实用工具。一个经典的点亮型适体系统是3,5-二氟-4-羟基亚苄基（DFHBI）荧光团与RNA适体菠菜素（Spinach）的组合，该系统已成功应用于细菌和哺乳动物细胞。然而，点亮型适体尚未在藻类中使用。在此，我们表明，一个携带菠菜素且转录融合到aphA-6基因的简单载体，可有效地用于在莱茵衣藻的叶绿体中产生功能性点亮型适体。用DFHBI孵育后，使用激光共聚焦显微镜观察表达aphA-6/菠菜素mRNA的品系，以评估点亮型适体在莱茵衣藻叶绿体中的功能。清晰且强烈的荧光以离散斑点的形式定位于叶绿体。缺乏菠菜素的菌株中没有背景荧光。点亮型适体可进一步改造用于藻类中RNA成像或开发基因编码生物传感器。
