Artificial OFF-Riboswitches That Downregulate Internal Ribosome Entry without Hybridization Switches in a Eukaryotic Cell-Free Translation System

Abstract

We constructed novel artificial riboswitches that function in a eukaryotic translation system (wheat germ extract), by rationally implanting an in vitro-selected aptamer into the intergenic internal ribosome entry site (IRES) of Plautia stali intestine virus. These eukaryotic OFF-riboswitches (OFF-eRSs) ligand-dose-dependently downregulate IRES-mediated translation without hybridization switches, which typical riboswitches utilize for gene regulation. The hybridization-switch-free mechanism not only allows for easy design but also requires less energy for regulation, resulting in a higher switching efficiency than hybridization-switch-based OFF-eRSs provide. In addition, even a small ligand such as theophylline can induce satisfactory repression, in contrast to other types of OFF-eRSs that modulate the 5' cap-dependent canonical translation. Because our proposed hybridization-switch-free OFF-eRSs are based on a versatile IRES that functions well in many types of eukaryotic translation systems, they would be widely usable elements for synthetic gene circuits in both cell-free and cell-based synthetic biology.

Riboswitches for Controlled Expression of Therapeutic Transgenes Delivered by Adeno-Associated Viral Vectors

“…This system enabled over 30-fold induction of eukaryotic gene expression. These constructs were later adapted into IRES-mediated off-switches [91], including off-switches in which IRES stem formation was controlled directly by aptamer binding rather than by binding-induced strand exchange with aIRES or aaIRES [92]. A 2020 follow-up publication reported several similar switches which were isolated using a strategy similar to that used to select paromomycin aptamers [86,93].…”

RNA-responsive elements for eukaryotic translational control

“…1b). Although IRESs have been adapted to sense small molecules 15 , IRES-based systems have not been designed to respond to trRNAs. We first selected IRES modules from viral databases and tested them in a human embryonic kidney 293 (HEK293) cell-based transfection assay (Extended Data Fig.…”

Synthetic small RNAs: Current status, challenges, and opportunities

“…Therefore, these riboswitches can find significant use as IRES‐based OFF riboswitches. Furthermore, these riboswitches were also found to function well in cell‐free translation systems of wheat germ extract …”

“…A theophylline-dependent riboswitch has been created for the regulation of translation in plastids of tobacco when theophylline is exogenously applied. 119,120 Over the last few decades, researchers have focused on RNAi technology for basic research, synthetic biology, and biomedicine. 118 Rational riboswitch constructs have been developed in eukaryotes for the downregulation of internal ribosome entry site (IRES)-mediated translation.…”

“…Furthermore, these riboswitches were also found to function well in cell-free translation systems of wheat germ extract. 119,120 Over the last few decades, researchers have focused on RNAi technology for basic research, synthetic biology, and biomedicine. Unfortunately, the constitutive knockdown of genes by RNAi may give undesired results, and this is a great problem in biomedical applications such as gene therapy.…”