Co-transcriptional production of programmable RNA condensates and synthetic organelles.

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.

Modular RNA motifs for orthogonal phase separated compartments.

Recent discoveries in biology have highlighted the importance of protein and RNA-based condensates as an alternative to classical membrane-bound organelles. Here, we demonstrate the design of pure RNA condensates from nanostructured, star-shaped RNA motifs. We generate condensates using two different RNA nanostar architectures: multi-stranded nanostars whose binding interactions are programmed via linear overhangs, and single-stranded nanostars whose interactions are programmed via kissing loops. Through systematic sequence design, we demonstrate that both architectures can produce orthogonal (distinct and immiscible) condensates, which can be individually tracked via fluorogenic aptamers. We also show that aptamers make it possible to recruit peptides and proteins to the condensates with high specificity. Successful co-transcriptional formation of condensates from single-stranded nanostars suggests that they may be genetically encoded and produced in living cells. We provide a library of orthogonal RNA condensates that can be modularly customized and offer a route toward creating systems of functional artificial organelles for the task of compartmentalizing molecules and biochemical reactions.

RNA nanotechnology on the horizon: Self-assembly, chemical modifications, and functional applications.

RNA nanotechnology harnesses the unique chemical and structural properties of RNA to build nanoassemblies and supramolecular structures with dynamic and functional capabilities. This review focuses on design and assembly approaches to building RNA structures, the RNA chemical modifications used to enhance stability and functionality, and modern-day applications in therapeutics, biosensing, and bioimaging.

Assembly of RNA Nanostructures from Double-Crossover Tiles.

Artificial self-assembling RNA scaffolds can be produced from many types of RNA motifs that are rationally designed. These scaffolds are of interest as nanoscale organizers, with applications in drug delivery and synthetic cells. Here we describe design strategies, production methods, and imaging of micrometer-sized RNA nanotubes and lattices that assemble from RNA tiles comprising multiple distinct strands.

Design and Characterization of RNA Nanotubes.

RNA is a functionally rich and diverse biomaterial responsible for regulating several cellular processes. This functionality has been harnessed to build predominately small nanoscale structures for drug delivery and the treatment of disease. The understanding of design principles to build large RNA structures will allow for further control of stoichiometry and spatial arrangement drugs and ligands. We present the design and characterization of RNA nanotubes that self-assemble from programmable monomers, or tiles, formed by five distinct RNA strands. Tiles include double crossover junctions and assemble via single-stranded sticky-end domains. We find that nanotube formation is dependent on the intertile crossover distance. The average length observed for the annealed RNA nanotubes is ≈1.5 µm, with many nanotubes exceeding 10 µm, enabling the characterization of RNA nanotubes length distribution via fluorescence microscopy. Assembled tubes were observed to be stable for more than 24 h, however post-annealing growth under isothermal conditions does not occur. Nanotubes assemble also from RNA tiles modified to include a single-stranded overhang (toehold), suggesting that it may be possible to decorate these large RNA scaffolds with nanoparticles or other nucleic acid molecules.

RNA Fibers as Optimized Nanoscaffolds for siRNA Coordination and Reduced Immunological Recognition.

RNA is a versatile biomaterial that can be used to engineer nanoassemblies for personalized treatment of various diseases. Despite promising advancements, the design of RNA nanoassemblies with minimal recognition by the immune system remains a major challenge. Here, an approach is reported to engineer RNA fibrous structures to operate as a customizable platform for efficient coordination of siRNAs and for maintaining low immunostimulation. Functional RNA fibers are studied in silico and their formation is confirmed by various experimental techniques and visualized by atomic force microscopy (AFM). It is demonstrated that the RNA fibers offer multiple advantages among which are: i) programmability and modular design that allow for simultaneous controlled delivery of multiple siRNAs and fluorophores, ii) reduced immunostimulation when compared to other programmable RNA nanoassemblies, and iii) simple production protocol for endotoxin-free fibers with the option of their cotranscriptional assembly. Furthermore, it is shown that functional RNA fibers can be efficiently delivered with various organic and inorganic carriers while retaining their structural integrity in cells. Specific gene silencing triggered by RNA fibers is assessed in human breast cancer and melanoma cell lines, with the confirmed ability of functional fibers to selectively target single nucleotide mutations.

Correction: Programmable RNA microstructures for coordinated delivery of siRNAs.

Correction for 'Programmable RNA microstructures for coordinated delivery of siRNAs' by Jaimie Marie Stewart et al., Nanoscale, 2016, 8, 17542-17550.

Self-assembly of multi-stranded RNA motifs into lattices and tubular structures.

Rational design of nucleic acid molecules yields self-assembling scaffolds with increasing complexity, size and functionality. It is an open question whether design methods tailored to build DNA nanostructures can be adapted to build RNA nanostructures with comparable features. Here we demonstrate the formation of RNA lattices and tubular assemblies from double crossover (DX) tiles, a canonical motif in DNA nanotechnology. Tubular structures can exceed 1 µm in length, suggesting that this DX motif can produce very robust lattices. Some of these tubes spontaneously form with left-handed chirality. We obtain assemblies by using two methods: a protocol where gel-extracted RNA strands are slowly annealed, and a one-pot transcription and anneal procedure. We identify the tile nick position as a structural requirement for lattice formation. Our results demonstrate that stable RNA structures can be obtained with design tools imported from DNA nanotechnology. These large assemblies could be potentially integrated with a variety of functional RNA motifs for drug or nanoparticle delivery, or for colocalization of cellular components.

Programmable RNA microstructures for coordinated delivery of siRNAs.

RNA is a natural multifunctional polymer, and is an essential component in both complex pathways and structures within the cellular environment. For this reason, artificial self-assembling RNA nanostructures are emerging as a powerful tool with broad applications in drug delivery and metabolic pathway regulation. To date, coordinated delivery of functional molecules via programmable RNA assemblies has been primarily done using nanosize RNA scaffolds. However, larger scaffolds could expand existing capabilities for spatial arrangement of ligands, and enable the controlled delivery of highly concentrated molecular loads. Here, we investigate whether micron-size RNA scaffolds can be assembled and further functionalized with different cargos (e.g. various siRNAs and fluorescent tags) for their synchronized delivery to diseased cells. Since known design approaches to build large RNA scaffolds are still underdeveloped, we apply a tiling method widely used in DNA nanotechnology. DNA tiles have been extensively used to build a variety of scalable and modular structures that are easily decorated with other ligands. Here, we adapt a double crossover (DX) DNA tile motif to design de novo DX RNA tiles that assemble and form lattices via programmed sticky end interactions. We optimize assembly protocols to guarantee high yield of RNA lattices. The resulting constructs are robust and modular with respect to the presence of distinct siRNAs and fluorophores. RNA tiles and lattices are successfully transfected in either human breast cancer or prostate cancer cells, where they efficiently knockdown the expression of target genes. Blood serum stability assays indicate that RNA lattices are more resilient to nuclease degradation when compared to individual tiles, thus making them better suited for therapeutic purposes. Overall, because of its design simplicity, we anticipate that this approach will be utilized for a wide range of applications in therapeutic RNA nanotechnology.