TY - JOUR
T1 - mRNA imaging in the chloroplast of Chlamydomonas reinhardtii using the light-up aptamer Spinach
AU - Guzmán-Zapata, Daniel
AU - Domínguez-Anaya, Yael
AU - Macedo-Osorio, Karla S.
AU - Tovar-Aguilar, Andrea
AU - Castrejón-Flores, José L.
AU - Durán-Figueroa, Noé V.
AU - Badillo-Corona, Jesús A.
N1 - Publisher Copyright:
© 2017 Elsevier B.V.
PY - 2017/6/10
Y1 - 2017/6/10
N2 - 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.
AB - 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.
KW - Chlamydomonas
KW - Chloroplast
KW - Light-up aptamer
KW - RNA imaging in vivo
UR - http://www.scopus.com/inward/record.url?scp=85018438614&partnerID=8YFLogxK
U2 - 10.1016/j.jbiotec.2017.03.028
DO - 10.1016/j.jbiotec.2017.03.028
M3 - Artículo
C2 - 28359866
SN - 0168-1656
VL - 251
SP - 186
EP - 188
JO - Journal of Biotechnology
JF - Journal of Biotechnology
ER -