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Molecular complexes composed of RNA molecules and proteins are promising multifunctional nanostructures for a wide variety of applications in biological cells or in artificial cellular systems. In this study, we systematically address some of the challenges associated with the expression and assembly of such hybrid structures using cell-free gene expression systems. As a model structure, we investigated a pRNA-derived RNA scaffold functionalized with four distinct aptamers, three of which bind to proteins, streptavidin and two fluorescent proteins, while one binds the small molecule dye malachite green (MG). Using MG fluorescence and Förster resonance energy transfer (FRET) between the RNA-scaffolded proteins, we assess critical assembly parameters such as chemical stability, binding efficiency, and also resource sharing effects within the reaction compartment. We then optimize simultaneous expression and coassembly of the RNA-protein nanostructure within a single-compartment cell-free gene expression system. We demonstrate expression and assembly of the multicomponent nanostructures inside of emulsion droplets and their aptamer-mediated localization onto streptavidin-coated substrates, plus the successful assembly of the hybrid structures inside of bacterial cells. [ABSTRACT FROM AUTHOR]

Natural stable RNAs fold and assemble into complex three-dimensional architectures by relying on the hierarchical formation of intricate, recurrent networks of noncovalent tertiary interactions. These sequence-dependent networks specify RNA structural modules enabling orientational and topological control of helical struts to form larger self-folding domains. Borrowing concepts from linguistics, we defined an extended structural syntax of RNA modules for programming RNA strands to assemble into complex, responsive nanostructures under both thermodynamic and kinetic control. Based on this syntax, various RNA building blocks promote the multimolecular assembly of objects with well-defined three-dimensional shapes as well as the isothermal folding of long RNAs into complex single-stranded nanostructures during transcription. This work offers a glimpse of the limitless potential of RNA as an informational medium for designing programmable and functional nanomaterials useful for synthetic biology, nanomedicine, and nanotechnology. [ABSTRACT FROM AUTHOR]

Current work reports the use of single-stranded RNA toeholds of different lengths to promote the reassociation of various RNA-DNA hybrids, which results in activation of multiple split functionalities inside human cells. The process of reassociation is analyzed and followed with a novel computational multistrand secondary structure prediction algorithm and various experiments. All of our previously designed RNA/DNA nanoparticles employed single-stranded DNA toeholds to initiate reassociation. The use of RNA toeholds is advantageous because of the simpler design rules, the shorter toeholds, and the smaller size of the resulting nanoparticles (by up to 120 nucleotides per particle) compared to the same hybrid nanoparticles with single-stranded DNA toeholds. Moreover, the cotranscriptional assemblies result in higher yields for hybrid nanoparticles with ssRNA toeholds. [ABSTRACT FROM AUTHOR]

We report a generalized methodology for the one-pot production of chemically modified functional RNA nanoparticles during in vitro transcription with T7 RNA polymerase. The efficiency of incorporation of 2′-fluoro-dNTP in the transcripts by the wild type T7 RNA polymerase dramatically increases in the presence of manganese ions, resulting in a high-yield production of chemically modified RNA nanoparticles functionalized with siRNAs that are resistant to nucleases from human blood serum. Moreover, the unpurified transcription mixture can be used for functional ex vivo pilot experiments. [ABSTRACT FROM AUTHOR]

Our recent advancements in RNA nanotechnology introduced novel nanoscaffolds (nanorings); however, the potential of their use for biomedical applications was never fully revealed. As presented here, besides functionalization with multiple different short interfering RNAs for combinatorial RNA interference (e.g., against multiple HIV-1 genes), nanorings also allow simultaneous embedment of assorted RNA aptamers, fluorescent dyes, proteins, as well as recently developed RNA-DNA hybrids aimed to conditionally activate multiple split functionalities inside cells.