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.Keywords: nanoparticles; nanostructures; RNA architectonics; RNA folding; RNA nanotechnology; RNA self-assembly; tectoRNAs;

Nevertheless, the synthetic RNA architectures achieved thus far consist largely of static, rigid particles that are still far from matching the structural and functional complexity of natural responsive structural elements such as the ribosome, large ribozymes, and riboswitches. Thus, the next step in synthetic RNA design will involve new ways to implement these same types of dynamic and responsive architectures into nanostructures functioning as real nanomachines in and outside the cell. RNA nanotechnology will likely garner broader utility and influence with a greater focus on the interplay between thermodynamic and kinetic influences on RNA self-assembly and using natural RNAs as guiding principles.

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.

RNA is an attractive biopolymer for nanodesign of self-assembling particles for nanobiotechnology and synthetic biology. Here, we experimentally characterize by biochemical and biophysical methods the formation of thermostable and ribonuclease resistant RNA nanorings previously proposed by computational design. High yields of fully programmable nanorings were produced based on several RNAI/IIi kissing complex variants selected for their ability to promote polygon self-assembly. This self-assembly strategy relying on the particular geometry of bended kissing complexes has potential for developing short interfering RNA delivery agents.

We detail a method originally described by Okahata et al. (Macromol. Rapid Commun. 2002, 23, 252−255) to prepare noncovalent self-assembling films by exchanging the counter-ions of the nucleic acid phosphate moieties with those of cationic lipid amphiphiles. We are able to control the strength and surface properties of these films by varying the composition between blends of DNA of high molecular weight and RNA of low molecular weight. X-ray and AFM results indicate that these films have a lamellar multilayered structure with layers of nucleic acid separated by layers of cationic amphiphile. The tensile strength of the blended films between DNA and RNA increases elastically with DNA content. The length as well as the molecular structure of nucleic acids can affect the topology and mechanical properties of these films. We suspect that the permeability properties of these films make them good candidates for further biological applications in vivo.

Abstract

One challenge in supramolecular chemistry is the design of versatile, self-assembling building blocks to attain total control of arrangement of matter at a molecular level. We have achieved reliable prediction and design of the three-dimensional structure of artificial RNA building blocks to generate molecular jigsaw puzzle units called tectosquares. They can be programmed with control over their geometry, topology, directionality, and addressability to algorithmically self-assemble into a variety of complex nanoscopic fabrics with predefined periodic and aperiodic patterns and finite dimensions. This work emphasizes the modular and hierarchical characteristics of RNA by showing that small RNA structural motifs can code the precise topology of large molecular architectures. It demonstrates that fully addressable materials based on RNA can be synthesized and provides insights into self-assembly processes involving large populations of RNA molecules.

The right angle (RA) motif, previously identified in the ribosome and used as a structural module for nano-construction, is a recurrent structural motif of 13 nucleotides that establishes a 90° bend between two adjacent helices. Comparative sequence analysis was used to explore the sequence space of the RA motif within ribosomal RNAs in order to define its canonical sequence space signature. We investigated the sequence constraints associated with the RA signature using several artificial self-assembly systems. Thermodynamic and topological investigations of sequence variants associated with the RA motif in both minimal and expanded structural contexts reveal that the presence of a helix at the 3′ end of the RA motif increases the thermodynamic stability and rigidity of the resulting three-helix junction domain. A search for the RA in naturally occurring RNAs as well as its experimental characterization led to the identification of the RA in groups IC1 and ID intron ribozymes, where it is suggested to play an integral role in stabilizing peripheral structural domains. The present study exemplifies the need of empirical analysis of RNA structural motifs for facilitating the rational design and structure prediction of RNAs.Graphical AbstractDownload high-res image (387KB)Download full-size imageResearch Highlights► The sequence space signature of the RA motif is determined by comparative structural and sequence analysis of ribosomal RNAs. ► RA biophysical and topological properties are characterized by self-assembly. ► Experimental and theoretical analyses support evidence of RA in group I introns. ► Data allow for predictive structural model of one of the peripheral elements of group I introns.

•We describe computational methodologies for designing RNA nanocubes.•3 different cube designs are computationally and experimentally characterized.•An Anisotropic Network Model is used to understand the dynamic nature of the cubes.•Computational results explain and predict the experimental outcomes.•Outcomes include cube sizes, melting temperatures and assembly yields.The fast-developing field of RNA nanotechnology requires the adoption and development of novel and faster computational approaches to modeling and characterization of RNA-based nano-objects. We report the first application of Elastic Network Modeling (ENM), a structure-based dynamics model, to RNA nanotechnology. With the use of an Anisotropic Network Model (ANM), a type of ENM, we characterize the dynamic behavior of non-compact, multi-stranded RNA-based nanocubes that can be used as nano-scale scaffolds carrying different functionalities. Modeling the nanocubes with our tool NanoTiler and exploring the dynamic characteristics of the models with ANM suggested relatively minor but important structural modifications that enhanced the assembly properties and thermodynamic stabilities. In silico and in vitro, we compared nanocubes having different numbers of base pairs per side, showing with both methods that the 10 bp-long helix design leads to more efficient assembly, as predicted computationally. We also explored the impact of different numbers of single-stranded nucleotide stretches at each of the cube corners and showed that cube flexibility simulations help explain the differences in the experimental assembly yields, as well as the measured nanomolecule sizes and melting temperatures. This original work paves the way for detailed computational analysis of the dynamic behavior of artificially designed multi-stranded RNA nanoparticles.Download high-res image (120KB)Download full-size image

The structural information encoding specific conformations of natural RNAs can be implemented within artificial RNA sequences to control both three-dimensional (3D) shape and self-assembling interfaces for nanotechnology and synthetic biology applications. We have identified three natural RNA motifs known to direct helical topology into approximately 90° bends: a five-way tRNA junction, a three-way junction, and a two-helix bend. These three motifs, embedded within rationally designed RNAs (tectoRNA), were chosen for generating square-shaped tetrameric RNA nanoparticles. The ability of each motif to direct the formation of supramolecular assemblies was compared by both native gel assays and atomic force microscopy. While there are multiple structural solutions for building square-shaped RNA particles, differences in the thermodynamics and molecular dynamics of the 90° motif can lead to different biophysical behaviors for the resulting supramolecular complexes. We demonstrate via structural assembly programming how the different 90° motifs can preferentially direct the formation of either 2D or 3D assemblies.