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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 G-quadruplexes (G4s) are well known non-canonical DNA secondary structures that can form in human cells. Most of the tools available to investigate G4-biology rely on small molecule ligands that stabilise these structures. However, the development of probes that disrupt G4s is equally important to study their biology. In this study, we investigated the disruption of G4s using Locked Nucleic Acids (LNA) as invader probes. We demonstrated that strategic positioning of LNA-modifications within short oligonucleotides (10 nts.) can significantly accelerate the rate of G4-disruption. Single-molecule experiments revealed that short LNA-probes can promote disruption of G4s with mechanical stability sufficient to stall polymerases. We corroborated this using a single-step extension assay, revealing that short LNA-probes can relieve replication dependent polymerase-stalling at G4 sites. We further demonstrated the potential of such LNA-based probes to study G4-biology in cells. By using a dual-luciferase assay, we found that short LNA probes can enhance the expression of c-KIT to levels similar to those observed when the c-KIT promoter is mutated to prevent the formation of the c-KIT1 G4. Collectively, our data suggest a potential use of rationally designed LNA-modified oligonucleotides as an accessible chemical-biology tool for disrupting individual G4s and interrogating their biological functions in cells.