Composing RNA Nanostructures from a Syntax of RNA Structural Modules

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

Fluorescent Monitoring of RNA Assembly and Processing Using the Split-Spinach Aptamer

As insights into RNA’s many diverse cellular roles continue to be gained, interest and applications in RNA self-assembly and dynamics remain at the forefront of structural biology. The bifurcation of functional molecules into nonfunctional fragments provides a useful strategy for controlling and monitoring cellular RNA processes and functionalities. Herein we present the bifurcation of the preexisting Spinach aptamer and demonstrate its utility as a novel split aptamer system for monitoring RNA self-assembly as well as the processing of pre-short interfering substrates. We show for the first time that the Spinach aptamer can be divided into two nonfunctional halves that, once assembled, restore the original fluorescent signal characteristic of the unabridged aptamer. In this regard, the split-Spinach aptamer is represented as a potential tool for monitoring the self-assembly of artificial and/or natural RNAs

Co-transcriptional Assembly of Chemically Modified RNA Nanoparticles Functionalized with siRNAs

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