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

Ortega, Alvaro Dario1,2; Takhaveev, Vakil1; Vedelaar, Silke Roelie1; Long, Yi3,4,8; Mestre-Farras, Neus1,9; Incarnato, Danny5; Ersoy, Franziska3,7; Olsen, Lars Folke4; Mayer, Gunter3,6; Heinemann, Matthias1

2021-11-18

10.1016/j.chembiol.2021.04.006

Cell Chemical Biology   影响因子和分区

2451-9448

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.

SCI

英语

其他

European Union[613745,289995,642738]

Biochemistry & Molecular Biology

Biochemistry & Molecular Biology

WOS:000722642500003

CELL PRESS

Web of Science

1.Univ Groningen, Groningen Biomol Sci & Biotechnol Inst, Mol Syst Biol, NL-9747 AG Groningen, Netherlands
2.Univ Complutense Madrid, Fac Biol, Dept Cell Biol, Madrid 28040, Spain
3.Univ Bonn, LIMES Inst, D-53121 Bonn, Germany
4.Univ Southern Denmark, Inst Biochem & Mol Biol, DK-5230 Odense M, Denmark
5.Univ Groningen, Groningen Biomol Sci & Biotechnol Inst, Mol Genet, NL-9747 AG Groningen, Netherlands
6.Univ Bonn, Ctr Aptamer Res & Dev, D-53121 Bonn, Germany
7.Leibniz Univ Hannover, Inst Food Chem, D-30167 Hannover, Germany
8.Southern Univ Sci & Technol Hosp, Med Res Ctr, Shenzhen 518055, Guangdong, Peoples R China
9.Barcelona Inst Sci & Technol, Ctr Genom Regulat CRG, Barcelona 08003, Spain

Ortega, Alvaro Dario,Takhaveev, Vakil,Vedelaar, Silke Roelie,et al. A synthetic RNA-based biosensor for fructose-1,6-bisphosphate that reports glycolytic flux[J]. Cell Chemical Biology,2021,28(11).

Ortega, Alvaro Dario.,Takhaveev, Vakil.,Vedelaar, Silke Roelie.,Long, Yi.,Mestre-Farras, Neus.,...&Heinemann, Matthias.(2021).A synthetic RNA-based biosensor for fructose-1,6-bisphosphate that reports glycolytic flux.Cell Chemical Biology,28(11).

Ortega, Alvaro Dario,et al."A synthetic RNA-based biosensor for fructose-1,6-bisphosphate that reports glycolytic flux".Cell Chemical Biology 28.11(2021).