Synthetic circular RNA switches and circuits that control protein expression in mammalian cells.

Synthetic messenger RNA (mRNA) has been focused on as an emerging application for mRNA-based therapies and vaccinations. Recently, synthetic circular RNAs (circRNAs) have shown promise as a new class of synthetic mRNA that enables superior stability and persistent gene expression in cells. However, translational control of circRNA remained challenging. Here, we develop 'circRNA switches' capable of controlling protein expression from circRNA by sensing intracellular RNA or proteins. We designed microRNA (miRNA) and protein-responsive circRNA switches by inserting miRNA-binding or protein-binding sequences into untranslated regions (UTRs), or Coxsackievirus B3 Internal Ribosome Entry Site (CVB3 IRES), respectively. Engineered circRNAs efficiently expressed reporter proteins without inducing severe cell cytotoxicity and immunogenicity, and responded to target miRNAs or proteins, controlling translation levels from circRNA in a cell type-specific manner. Moreover, we constructed circRNA-based gene circuits that selectively activated translation by detecting endogenous miRNA, by connecting miRNA and protein-responsive circRNAs. The designed circRNA circuits performed better than the linear mRNA-based circuits in terms of persistent expression levels. Synthetic circRNA devices provide new insights into RNA engineering and have a potential for RNA synthetic biology and therapies.

N 1-Methylpseudouridine substitution enhances the performance of synthetic mRNA switches in cells.

Synthetic messenger RNA (mRNA) tools often use pseudouridine and 5-methyl cytidine as substitutions for uridine and cytidine to avoid the immune response and cytotoxicity induced by introducing mRNA into cells. However, the influence of base modifications on the functionality of the RNA tools is poorly understood. Here we show that synthetic mRNA switches containing N1-methylpseudouridine (m1Ψ) as a substitution of uridine substantially out-performed all other modified bases studied, exhibiting enhanced microRNA and protein sensitivity, better cell-type separation ability, and comparably low immune stimulation. We found that the observed phenomena stem from the high protein expression from m1Ψ containing mRNA and efficient translational repression in the presence of target microRNAs or proteins. In addition, synthetic gene circuits with m1Ψ significantly improve performance in cells. These findings indicate that synthetic mRNAs with m1Ψ modification have enormous potentials in the research and application of biofunctional RNA tools.

Thermotaxis in Chlamydomonas is brought about by membrane excitation and controlled by redox conditions.

Temperature is physiologically critical for all living organisms, which cope with temperature stress using metabolic and behavioral responses. In unicellular and some multicellular organisms, thermotaxis is a behavioral response to avoid stressful thermal environments and promote accumulation in an optimal thermal environment. In this study, we examined whether Chlamydomonas reinhardtii, a unicellular green alga, demonstrated thermotaxis. We found that between 10 °C and 30 °C, Chlamydomonas cells migrated toward lower temperatures independent of cultivation temperature. Interestingly, when we applied reagents to change intracellular reduction-oxidation (redox) conditions, we saw that thermotaxis was enhanced, suppressed, or reversed, depending on the redox conditions and cultivation temperature. Thermotaxis was almost absent in ppr2 and ppr3 mutants, which cannot swim backward because of a defect in generating calcium current in flagella. The frequency of spontaneous backward swimming was lower at more favorable temperature, suggesting a pivotal role of spontaneous backward swimming generated by flagellar membrane excitation.