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Condensation of RNA and proteins is central to cellular functions, and the ability to program it would be valuable in synthetic biology and synthetic cell science. Here we introduce a modular platform for engineering synthetic RNA condensates from tailor-made, branched RNA nanostructures that fold and assemble co-transcriptionally. Up to three orthogonal condensates can form simultaneously and selectively accumulate fluorophores through embedded fluorescent light-up aptamers. The RNA condensates can be expressed within synthetic cells to produce membrane-less organelles with a controlled number and relative size, and showing the ability to capture proteins using selective protein-binding aptamers. The affinity between otherwise orthogonal nanostructures can be modulated by introducing dedicated linker constructs, enabling the production of bi-phasic RNA condensates with a prescribed degree of interphase mixing and diverse morphologies. The in situ expression of programmable RNA condensates could underpin the spatial organization of functionalities in both biological and synthetic cells. 

Abstract We present a new class of DNA‐based nanoswitches that, upon enzymatic repair, could undergo a conformational change mechanism leading to a change in fluorescent signal. Such folding‐upon‐repair DNA nanoswitches are synthetic DNA sequences containingO⁶‐methyl‐guanine (O⁶‐MeG) nucleobases and labelled with a fluorophore/quencher optical pair. The nanoswitches are rationally designed so that only upon enzymatic demethylation of theO⁶‐MeG nucleobases they can form stable intramolecular Hoogsteen interactions and fold into an optically active triplex DNA structure. We have first characterized the folding mechanism induced by the enzymatic repair activity through fluorescent experiments and Molecular Dynamics simulations. We then demonstrated that the folding‐upon‐repair DNA nanoswitches are suitable and specific substrates for different methyltransferase enzymes including the human homologue (hMGMT) and they allow the screening of novel potential methyltransferase inhibitors.