RT Journal Article
T1 A synthetic RNA-based biosensor for fructose-1,6-bisphosphatethat reports glycolytic flux
A1 Ortega Moreno, Álvaro Dario
A1 Takhaveev, Vakil
A1 Vedelaar, Silke Roelie
A1 Long, Yi
A1 Mestre-Farràs, Neus
A1 Incarnato, Danny
A1 Ersoy Karaçuha, Franziska
A1 Olsen, Lars Folke
A1 Mayer, Günter
A1 Heinemann, Matthias
AB RNA-based sensors for intracellular metabolites are a promising solution to the emerging issue of metabolic heterogeneity. However, their development, i.e., the conversion of an aptamer into an in vivo-functional intracellular metabolite sensor, still harbors challenges. Here, we accomplished this for the glycolytic fluxsignaling metabolite, fructose-1,6-bisphosphate (FBP). Starting from in vitro selection of an aptamer, we constructed device libraries with a hammerhead ribozyme as actuator. Using high-throughput screening in yeast with fluorescence-activated cell sorting (FACS), next-generation sequencing, and genetic-environmental perturbations to modulate the intracellular FBP levels, we identified a sensor that generates ratiometric fluorescent readout. An abrogated response in sensor mutants and occurrence of two sensor conformations— revealed by RNA structural probing—indicated in vivo riboswitching activity. Microscopy showed that the sensor can differentiate cells with different glycolytic fluxes within yeast populations, opening research avenues into metabolic heterogeneity. We demonstrate the possibility to generate RNA-based sensors for intracellular metabolites for which no natural metabolite-binding RNA element exits.
PB Elsevier
SN Electronic: 2451-9448
YR 2021
FD 2021-04-28
LK https://hdl.handle.net/20.500.14352/8578
UL https://hdl.handle.net/20.500.14352/8578
LA eng
NO Horizonte 2020 Unión Europea
NO Unión Europea FP7
DS Docta Complutense
RD 4 abr 2025