A synthetic RNA-based biosensor for fructose-1,6-bisphosphate that reports glycolytic flux

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Ortega Moreno, Álvaro Dario and Takhaveev, Vakil and Vedelaar, Silke Roelie and Long, Yi and Mestre-Farràs, Neus and Incarnato, Danny and Ersoy Karaçuha, Franziska and Olsen, Lars Folke and Mayer, Günter and Heinemann, Matthias (2021) A synthetic RNA-based biosensor for fructose-1,6-bisphosphate that reports glycolytic flux. Cell Chemical Biology, 28 (11). pp. 1-15. ISSN Electronic: 2451-9448

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Official URL: https://doi.org/10.1016/j.chembiol.2021.04.006



Abstract

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.


Item Type:Article
Uncontrolled Keywords:Aptamer; Biosensor; Ribozyme; Screening; Fructose-1,6-bisphosphate; Metabolic heterogeneity; Glycolysis; RNA
Subjects:Medical sciences > Biology > Cytology
Medical sciences > Biology > Molecular biology
Medical sciences > Biology > Genetics
ID Code:69199
Deposited On:14 Dec 2021 15:19
Last Modified:05 Apr 2022 08:42

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