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

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 conformationsrevealed 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.

Real-Time Monitoring of the Yeast Intracellular State During Bioprocesses With a Toolbox of Biosensors

“…In many fermentation processes, end products are synthesized starting from sugars (Francois et al, 2020;Maicas, 2020;Sharma et al, 2020). GlyRNA (Glycolytic RNA probe) was selected as an aptameric sensor for glycolytic flux, because degradation of its mRNA is sensible to the intracellular concentration of fructosebisphosphate (Ortega et al, 2021). Given that the sensor's response decreases with an increasing concentration of fructosebisphosphate, a negative peak denotes maximum glycolytic flux.…”

Organelle transporters and inter-organelle communication as drivers of metabolic regulation and cellular homeostasis

“…Proximity labeling with intact and split promiscuous biotin ligase (BirA) promise to uncover new tethering factors and metabolite transporters that selectively operate at MCSs [ 168 , 169 ]. Technologies such as in situ mass spectrometry imaging, isotopic labelling and fluorescent or genetically encoded RNA-based metabolite sensors will help reveal the flux of polar metabolites between different organelles [ [170] , [171] , [172] ]. Combined with functional genomics, in vitro reconstitution, and organelle-specific proteomics in different cells and tissues [ 173 , 174 ], these in situ labeling techniques may lead to the discovery of new pathways, or branches of known pathways, that ensure coordination of organelle function across a range of physiological states.…”

Generative and interpretable machine learning for aptamer design and analysis of in vitro sequence selection

“…After having established that the RBM log-likelihoods and the fitnesses of the aptamers in our dataset are strongly interrelated, we now use the RBM model to generate new sequences in silico (see Methods Sec Restricted Boltzmann Machine: Definition, training, sampling). Note that the number of available sequences at any round, <10 6 , is much smaller than the number of possible sequences over 20 nucleotides, 4 20 ' 10 12 . Hence, it is a non trivial problem to (where w μi is the weight of the connection between hidden unit μ and visible unit i for nucleotide s i ) to the corresponding hidden units for the sequences s in the dataset (gray) and average activity (black).…”

“…Multiple different experimental approaches exist to select specific molecular target binder such as antibodies, short peptides, proteins or small molecules. Single stranded oligonucleotides (DNA or RNA) have also been shown to be able to specifically bind with high affinity to a plethora of various targets, including small metabolites, proteins, nucleic acids, viruses, exosomes, and cells of specific tissue [1][2][3][4][5][6][7][8][9][10], showing promise for applications that range from diagnostics to targeted disease therapy [11]. These short oligonucleotides, called aptamers, are selected from an initial pool of sequences by a procedure known as Systematic Evolution of Ligands by Exponential Enrichment (SELEX) [12,13].…”