The future of RNA origami writ large Researchers have long fabricated intricate nanostructures from carefully linked DNA strands. Now they can use RNA made by gene expression, which avoids the costly strand synthesis and lengthy annealing steps necessary with DNA origami. Geary et al. used molecular modeling to extend the size of folded RNA origami structures (see the Perspective by Leontis and Westhof). The modeling revealed assembly patterns for linking single-stranded RNA into A-form helices. The authors created two-dimensional structures as large as 660 nucleotides on mica surfaces. Science , this issue p. 799 ; see also p. 732

Specific recognitions of GNRA tetraloops by small helical receptors are among the most widespread long-range packing interactions in large ribozymes. However, in contrast to GYRA and GAAA tetraloops, very few GNRA/receptor interactions have yet been identified to involve GGAA tetraloops in nature. A novel in vitro selection scheme based on a rigid self-assembling tectoRNA scaffold designed for isolation of intermolecular interactions with A-minor motifs has yielded new GGAA tetraloop-binding receptors with affinity in the nanomolar range. One of the selected receptors is a novel 12 nt RNA motif, (CCUGUG … AUCUGG), that recognizes GGAA tetraloop hairpin with a remarkable specificity and affinity. Its physical and chemical characteristics are comparable to those of the well-studied ‘11nt’ GAAA tetraloop receptor motif. A second less specific motif (CCCAGCCC … GAUAGGG) binds GGRA tetraloops and appears to be related to group IC3 tetraloop receptors. Mutational, thermodynamic and comparative structural analysis suggests that natural and in vitro selected GNRA receptors can essentially be grouped in two major classes of GNRA binders. New insights about the evolution, recognition and structural modularity of GNRA and A-minor RNA–RNA interactions are proposed.

Supra-molecular assembly is a powerful strategy used by nature for building nano-scale architectures with predefined sizes and shapes. Numerous challenges remain however to be solved in order to demonstrate precise control over the synthesis, folding and assembly of rationally designed three-dimensional (3D) nano-objects made of RNA. Using the transfer RNA molecule as a structural building block, we report the design, efficient synthesis and structural characterization of stable, modular 3D particles adopting the polyhedral geometry of a non-uniform square antiprism. The spatial control within the final architecture allows precise positioning and encapsulation of proteins. This work demonstrates that a remarkable degree of structural control can be achieved with RNA structural motifs to build thermostable 3D nano-architectures that do not rely on helix bundles or tensegrity. RNA 3D particles can potentially be used as carriers or scaffolds in nano-medicine and synthetic biology.

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 (NPs). The ability of each motif to direct the formation of supramolecular assemblies was compared by both native gel assays and atomic force microscopy (AFM). 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.

Stable RNAs are modular and hierarchical 3D architectures taking advantage of recurrent structural motifs to form extensive non-covalent tertiary interactions. Sequence and atomic structure analysis has revealed a novel submotif involving a minimal set of five nucleotides, termed the UA_handle motif (5′XU/ANnX3′). It consists of a U:A Watson–Crick: Hoogsteen trans base pair stacked over a classic Watson–Crick base pair, and a bulge of one or more nucleotides that can act as a handle for making different types of long-range interactions. This motif is one of the most versatile building blocks identified in stable RNAs. It enters into the composition of numerous recurrent motifs of greater structural complexity such as the T-loop, the 11-nt receptor, the UAA/GAN and the G-ribo motifs. Several structural principles pertaining to RNA motifs are derived from our analysis. A limited set of basic submotifs can account for the formation of most structural motifs uncovered in ribosomal and stable RNAs. Structural motifs can act as structural scaffoldings and be functionally and topologically equivalent despite sequence and structural differences. The sequence network resulting from the structural relationships shared by these RNA motifs can be used as a proto-language for assisting prediction and rational design of RNA tertiary structures.

RNA molecules take advantage of prevalent structural motifs to fold and assemble into well-defined 3D architectures. The A-minor junction is a class of RNA motifs that specifically controls coaxial stacking of helices in natural RNAs. A sensitive self-assembling supra-molecular system was used as an assay to compare several natural and previously unidentified A-minor junctions by native polyacrylamide gel electrophoresis and atomic force microscopy. This class of modular motifs follows a topological rule that can accommodate a variety of interchangeable A-minor interactions with distinct local structural motifs. Overall, two different types of A-minor junctions can be distinguished based on their functional self-assembling behavior: one group makes use of triloops or GNRA and GNRA-like loops assembling with helices, while the other takes advantage of more complex tertiary receptors specific for the loop to gain higher stability. This study demonstrates how different structural motifs of RNA can contribute to the formation of topologically equivalent helical stacks. It also exemplifies the need of classifying RNA motifs based on their tertiary structural features rather than secondary structural features. The A-minor junction rule can be used to facilitate tertiary structure prediction of RNAs and rational design of RNA parts for nanobiotechnology and synthetic biology.

RNA origami is a framework for the modular design of nanoscaffolds that can be folded from a single strand of RNA, and used to organize molecular components with nanoscale precision. Design of genetically expressible RNA origami, which must cotranscriptionally fold, requires modeling and design tools that simultaneously consider thermodynamics, folding pathway, sequence constraints, and pseudoknot optimization. Here, we describe RNA Origami Automated Design software (ROAD), which builds origami models from a library of structural modules, identifies potential folding barriers, and designs optimized sequences. Using ROAD, we extend the scale and functional diversity of RNA scaffolds, creating 32 designs of up to 2360 nucleotides, five that scaffold two proteins, and seven that scaffold two small molecules at precise distances. Micrographic and chromatographic comparison of optimized and nonoptimized structures validates that our principles for strand routing and sequence design substantially improve yield. By providing efficient design of RNA origami, ROAD may simplify construction of custom RNA scaffolds for nanomedicine and synthetic biology.

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.