title: mRNA imaging in the chloroplast of Chlamydomonas reinhardtii using the light-up aptamer Spinach.
abstract: 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.
authors.affiliation: Instituto Politécnico Nacional, Unidad Profesional Interdisciplinaria de Biotecnología, Av. Acueducto S/N., Col. Barrio La Laguna Ticomán, 07340 México City, Mexico.
authors.forename: Daniel
authors.initials: D
authors.lastname: Guzmán-Zapata
authors.affiliation: Instituto Politécnico Nacional, Unidad Profesional Interdisciplinaria de Biotecnología, Av. Acueducto S/N., Col. Barrio La Laguna Ticomán, 07340 México City, Mexico.
authors.forename: Yael
authors.initials: Y
authors.lastname: Domínguez-Anaya
authors.affiliation: Instituto Politécnico Nacional, Unidad Profesional Interdisciplinaria de Biotecnología, Av. Acueducto S/N., Col. Barrio La Laguna Ticomán, 07340 México City, Mexico.
authors.forename: Karla S
authors.initials: KS
authors.lastname: Macedo-Osorio
authors.affiliation: Instituto Politécnico Nacional, Unidad Profesional Interdisciplinaria de Biotecnología, Av. Acueducto S/N., Col. Barrio La Laguna Ticomán, 07340 México City, Mexico.
authors.forename: Andrea
authors.initials: A
authors.lastname: Tovar-Aguilar
authors.affiliation: Instituto Politécnico Nacional, Unidad Profesional Interdisciplinaria de Biotecnología, Av. Acueducto S/N., Col. Barrio La Laguna Ticomán, 07340 México City, Mexico.
authors.forename: José L
authors.initials: JL
authors.lastname: Castrejón-Flores
authors.affiliation: Instituto Politécnico Nacional, Unidad Profesional Interdisciplinaria de Biotecnología, Av. Acueducto S/N., Col. Barrio La Laguna Ticomán, 07340 México City, Mexico. Electronic address: nduranf@ipn.mx.
authors.forename: Noé V
authors.initials: NV
authors.lastname: Durán-Figueroa
authors.affiliation: Instituto Politécnico Nacional, Unidad Profesional Interdisciplinaria de Biotecnología, Av. Acueducto S/N., Col. Barrio La Laguna Ticomán, 07340 México City, Mexico. Electronic address: jbadilloc@ipn.mx.
authors.forename: Jesús A
authors.initials: JA
authors.lastname: Badillo-Corona
mesh_terms: D052157:Aptamers, Nucleotide / Q000235:genetics*; D001593:Benzyl Compounds; D016825:Chlamydomonas reinhardtii / Q000235:genetics*; D002736:Chloroplasts / Q000235:genetics*; D005453:Fluorescence; D005456:Fluorescent Dyes; D048288:Imidazolines; D019868:Kanamycin Kinase / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D018749:RNA, Plant / Q000235:genetics
chemical_list: C560120:3,5-difluoro-4-hydroxybenzylidene imidazolinone; D052157:Aptamers, Nucleotide; D001593:Benzyl Compounds; D005456:Fluorescent Dyes; D048288:Imidazolines; D012333:RNA, Messenger; D018749:RNA, Plant; D019868:Kanamycin Kinase
keywords: Chlamydomonas; Chloroplast; Light-up aptamer; RNA imaging in vivo