CE44 - Biochimie du Vivant

Harnessing RNA helicase activity for synthetic (ribo)regulation – HELISWITCH

Submission summary

Synthetic biology (SynBio) aims at the rational engineering of new biological functions or systems and their purposeful applications in industry, agriculture, environmental monitoring, defense, and healthcare. Synbio is a rapidly expanding segment of the bioeconomy with a global market share already valued at several € billions.

Several SynBio applications require biological devices that can be easily reprogrammed to detect new chemical or biological molecules of interest (e.g. biomarkers, drugs, environmental hazards), and convert their detection into a macroscopic observable of interest (e.g. reporter gene fluorescence, cell motility). Yet, despite recent advances, scalable ligand sensing platforms are critically lacking in the field, greatly hindering the advancement of many promising applications.

The HELISWITCH project aims to provide breakthrough solutions to this critical SynBio bottleneck. Here, we will deliver a new class of programmable ligand-responsive nucleic acid devices harnessing the biochemical or biological functions of helicases. These enzymes are involved in all cellular processes dealing with nucleic acids, including the major steps of gene expression. For instance, the bacterial Rho helicase terminates transcription by dissociating transcriptional complexes at specific genomic loci, while the eukaryotic Upf1 helicase triggers mRNA decay in a translation-dependent manner. In addition, helicases such as Rho or Upf1 display robust ATP-dependent molecular motor activity, a feature that can also be exploited advantageously to generate relevant signal outputs in vitro. Finally, some helicases like Upf1 are promiscuous and indifferently use DNA or RNA as substrates, increasing the flexibility and the range of potential SynBio applications.

Using Rho and Upf1 as model enzymes, we will demonstrate that the unique features of helicases constitute a decisive advantage to resolve the key issue of sensing scalability. Our work is based on an innovative technology workflow enabling the selection of ligand-responsive helicase switches (Heliswitches) that we already validated to produce a serotonin responsive reporter. We will first automate this workflow and expand its scope to achieve swift generation of on-demand heliswitches. Notably, we will develop automated procedures to create ON or OFF, Rho- or Upf1-dependent, and RNA or DNA switches. Using these procedures and three model small-molecule ligands of pharmacological interest (serotonin, kynurenine, cortisol), we will engineer a collection of reporter devices that work in vitro or in vivo, in prokaryotes or eukaryotes, and rely on different nucleic acid backbones or mechanisms, thereby illustrating our workflow capability and its robustness. Then, we will translate the unique features of these ligand-responsive devices into representative, proof-of-concept SynBio applications in vitro, in vivo, and with clinical samples. We will use serotonin-inducible heliswitches coupled to fluorescent reporters to screen libraries of tryptophan decarboxylase mutants in search of enzymes readily converting a non-cognate substrate (5-Hydroxytryptophan) into serotonin. We will also develop proof-of-concept in vitro biosensors based on helicase-controlled duplex unwinding or cell-free transcription-translation and use these heliswitch sensors to monitor the level of cortisol, a relevant marker for monitoring depression and suicidal relapse, in clinical samples from psychiatric patients.

Overall, we expect HELISWITCH to provide unique tools to program cellular behavior and advance the portable biosensor technologies critically needed in medical diagnostics and other key field applications.

Project coordination

Marc Boudvillain (Centre de biophysique moléculaire)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

CBM Centre de biophysique moléculaire
CBS Centre de biochimie structurale
CBS Centre de biochimie structurale

Help of the ANR 560,164 euros
Beginning and duration of the scientific project: December 2019 - 48 Months

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