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

Alvaro Darío Ortega*, Vakil Takhaveev, Silke Roelie Vedelaar, Yi Long, Neus Mestre-Farràs, Danny Incarnato, Franziska Ersoy, Lars Folke Olsen, Günter Mayer, Matthias Heinemann*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

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 flux-signaling 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.

Original languageEnglish
Article numberj.chembiol.2021.04.006
JournalCell Chemical Biology
DOIs
Publication statusE-pub ahead of print - 22-Apr-2021

Keywords

  • aptamer
  • biosensor
  • ribozyme
  • screening
  • fructose-1,6-bisphosphate
  • metabolic heterogeneity
  • glycolysis
  • RNA

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