In vitro selected GUAA tetraloop-binding receptors with structural plasticity and evolvability towards natural RNA structural modules.

GNRA tetraloop-binding receptor interactions are key components in the macromolecular assembly of a variety of functional RNAs. In nature, there is an apparent bias for GAAA/11nt receptor and GYRA/helix interactions, with the former interaction being thermodynamically more stable than the latter. While past in vitro selections allowed isolation of novel GGAA and GUGA receptors, we report herein an in vitro selection that revealed several novel classes of specific GUAA receptors with binding affinities comparable to those from natural GAAA/11nt interactions. These GUAA receptors have structural homology with double-locked bulge RNA modules naturally occurring in ribosomal RNAs. They display mutational robustness that enables exploration of the sequence/phenotypic space associated to GNRA/receptor interactions through epistasis. Their thermodynamic self-assembly fitness landscape is characterized by a rugged neutral network with possible evolutionary trajectories toward natural GNRA/receptor interactions. High throughput sequencing analysis revealed synergetic mutations located away from the tertiary interactions that positively contribute to assembly fitness. Our study suggests that the repertoire of GNRA/receptor interactions is much larger than initially thought from the analysis of natural stable RNA molecules and also provides clues for their evolution towards natural GNRA/receptors.

Ribosome engineering reveals the importance of 5S rRNA autonomy for ribosome assembly.

5S rRNA is an indispensable component of cytoplasmic ribosomes in all species. The functions of 5S rRNA and the reasons for its evolutionary preservation as an independent molecule remain unclear. Here we used ribosome engineering to investigate whether 5S rRNA autonomy is critical for ribosome function and cell survival. By linking circularly permutated 5S rRNA with 23S rRNA we generated a bacterial strain devoid of free 5S rRNA. Viability of the engineered cells demonstrates that autonomous 5S rRNA is dispensable for cell growth under standard conditions and is unlikely to have essential functions outside the ribosome. The fully assembled ribosomes carrying 23S-5S rRNA are highly active in translation. However, the engineered cells accumulate aberrant 50S subunits unable to form stable 70S ribosomes. Cryo-EM analysis revealed a malformed peptidyl transferase center in the misassembled 50S subunits. Our results argue that the autonomy of 5S rRNA is preserved due to its role in ribosome biogenesis.

Truncated tetrahedral RNA nanostructures exhibit enhanced features for delivery of RNAi substrates.

Using RNA as a material for nanoparticle construction provides control over particle size and shape at the nano-scale. RNA nano-architectures have shown promise as delivery vehicles for RNA interference (RNAi) substrates, allowing multiple functional entities to be combined on a single particle in a programmable fashion. Rather than employing a completely bottom-up approach to scaffold design, here multiple copies of an existing synthetic supramolecular RNA nano-architecture serve as building blocks along with additional motifs for the design of a novel truncated tetrahedral RNA scaffold, demonstrating that rationally designed RNA assemblies can themselves serve as modular pieces in the construction of larger rationally designed structures. The resulting tetrahedral scaffold displays enhanced characteristics for RNAi-substrate delivery in comparison to similar RNA-based scaffolds, as evidenced by its increased functional capacity, increased cellular uptake and ultimately an increased RNAi efficacy of its adorned Dicer substrate siRNAs. The unique truncated tetrahedral shape of the nanoparticle core appears to contribute to this particle's enhanced function, indicating the physical characteristics of RNA scaffolds merit significant consideration when designing platforms for delivery of functional RNAs via RNA nanoparticles.

Secondary Structural Model of Human MALAT1 Reveals Multiple Structure-Function Relationships.

Human metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is an abundant nuclear-localized long noncoding RNA (lncRNA) that has significant roles in cancer. While the interacting partners and evolutionary sequence conservation of MALAT1 have been examined, much of the structure of MALAT1 is unknown. Here, we propose a hypothetical secondary structural model for 8425 nucleotides of human MALAT1 using three experimental datasets that probed RNA structures in vitro and in various human cell lines. Our model indicates that approximately half of human MALAT1 is structured, forming 194 helices, 13 pseudoknots, five structured tetraloops, nine structured internal loops, and 13 intramolecular long-range interactions that give rise to several multiway junctions. Evolutionary conservation and covariation analyses support 153 of 194 helices in 51 mammalian MALAT1 homologs and 42 of 194 helices in 53 vertebrate MALAT1 homologs, thereby identifying an evolutionarily conserved core that likely has important functional roles in mammals and vertebrates. Data mining revealed that RNA modifications, somatic cancer-associated mutations, and single-nucleotide polymorphisms may induce structural rearrangements that sequester or expose binding sites for several cancer-associated microRNAs. Our findings reveal new mechanistic leads into the roles of MALAT1 by identifying several intriguing structure-function relationships in which the dynamic structure of MALAT1 underlies its biological functions.

Responsive self-assembly of tectoRNAs with loop-receptor interactions from the tetrahydrofolate (THF) riboswitch.

Naturally occurring RNAs are known to exhibit a high degree of modularity, whereby specific structural modules (or motifs) can be mixed and matched to create new molecular architectures. The modular nature of RNA also affords researchers the ability to characterize individual structural elements in controlled synthetic contexts in order to gain new and critical insights into their particular structural features and overall performance. Here, we characterized the binding affinity of a unique loop-receptor interaction found in the tetrahydrofolate (THF) riboswitch using rationally designed self-assembling tectoRNAs. Our work suggests that the THF loop-receptor interaction has been fine-tuned for its particular role as a riboswitch component. We also demonstrate that the thermodynamic stability of this interaction can be modulated by the presence of folinic acid, which induces a local structural change at the level of the loop-receptor. This corroborates the existence of a THF binding site within this tertiary module and paves the way for its potential use as a THF responsive module for RNA nanotechnology and synthetic biology.

Optimization of the Split-Spinach Aptamer for Monitoring Nanoparticle Assembly Involving Multiple Contiguous RNAs.

The fact that structural RNA motifs can direct RNAs to fold and self-assemble into predictable pre-defined structures is an attractive quality and driving force for RNA's use in nanotechnology. RNA's recognized diversity concerning cellular and synthetically selected functionalities, however, help explain why it continues to draw attention for new nano-applications. Herein, we report the modification of a bifurcated reporter system based on the previously documented Spinach aptamer/DFHBI fluorophore pair that affords the ability to confirm the assembly of contiguous RNA strands within the context of the previously reported multi-stranded RNA nanoring. Exploration of the sequence space associated with the base pairs flanking the aptamer core demonstrate that fluorescent feedback can be optimized to minimize the fluorescence associated with partially-assembled RNA nanorings. Finally, we demonstrate that the aptamer-integrated nanoring is capable of assembling directly from transcribed DNA in one pot.

Deducing putative ancestral forms of GNRA/receptor interactions from the ribosome.

Stable RNAs rely on a vast repertoire of long-range interactions to assist in the folding of complex cellular machineries such as the ribosome. The universally conserved L39/H89 interaction is a long-range GNRA-like/receptor interaction localized in proximity to the peptidyl transferase center of the large subunit of the ribosome. Because of its central location, L39/H89 likely originated at an early evolutionary stage of the ribosome and played a significant role in its early function. However, L39/H89 self-assembly is impaired outside the ribosomal context. Herein, we demonstrate that structural modularity principles can be used to re-engineer L39/H89 to self-assemble in vitro. The new versions of L39/H89 improve affinity and loop selectivity by several orders of magnitude and retain the structural and functional features of their natural counterparts. These versions of L39/H89 are proposed to be ancestral forms of L39/H89 that were capable of assembling and folding independently from proteins and post-transcriptional modifications. This work demonstrates that novel RNA modules can be rationally designed by taking advantage of the modular syntax of RNA. It offers the prospect of creating new biochemical models of the ancestral ribosome and increases the tool kit for RNA nanotechnology and synthetic biology.

A metastable rRNA junction essential for bacterial 30S biogenesis.

Tertiary sequence motifs encode interactions between RNA helices that create the three-dimensional structures of ribosomal subunits. A Right Angle motif at the junction between 16S helices 5 and 6 (J5/6) is universally conserved amongst small subunit rRNAs and forms a stable right angle in minimal RNAs. J5/6 does not form a right angle in the mature ribosome, suggesting that this motif encodes a metastable structure needed for ribosome biogenesis. In this study, J5/6 mutations block 30S ribosome assembly and 16S maturation in Escherichia coli. Folding assays and in-cell X-ray footprinting showed that J5/6 mutations favor an assembly intermediate of the 16S 5' domain and prevent formation of the central pseudoknot. Quantitative mass spectrometry revealed that mutant pre-30S ribosomes lack protein uS12 and are depleted in proteins uS5 and uS2. Together, these results show that impaired folding of the J5/6 right angle prevents the establishment of inter-domain interactions, resulting in global collapse of the 30S structure observed in electron micrographs of mutant pre-30S ribosomes. We propose that the J5/6 motif is part of a spine of RNA helices that switch conformation at distinct stages of assembly, linking peripheral domains with the 30S active site to ensure the integrity of 30S biogenesis.

Blind prediction of noncanonical RNA structure at atomic accuracy.

Prediction of RNA structure from nucleotide sequence remains an unsolved grand challenge of biochemistry and requires distinct concepts from protein structure prediction. Despite extensive algorithmic development in recent years, modeling of noncanonical base pairs of new RNA structural motifs has not been achieved in blind challenges. We report a stepwise Monte Carlo (SWM) method with a unique add-and-delete move set that enables predictions of noncanonical base pairs of complex RNA structures. A benchmark of 82 diverse motifs establishes the method's general ability to recover noncanonical pairs ab initio, including multistrand motifs that have been refractory to prior approaches. In a blind challenge, SWM models predicted nucleotide-resolution chemical mapping and compensatory mutagenesis experiments for three in vitro selected tetraloop/receptors with previously unsolved structures (C7.2, C7.10, and R1). As a final test, SWM blindly and correctly predicted all noncanonical pairs of a Zika virus double pseudoknot during a recent community-wide RNA-Puzzle. Stepwise structure formation, as encoded in the SWM method, enables modeling of noncanonical RNA structure in a variety of previously intractable problems.

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.

Supported Fluid Lipid Bilayer as a Scaffold to Direct Assembly of RNA Nanostructures.

RNA architectonics offers the possibility to design and assemble RNA into specific shapes, such as nanoscale 3D solids or nanogrids. Combining the minute size of these programmable shapes with precise positioning on a surface further enhances their potential as building blocks in nanotechnology and nanomedicine. Here we describe a bottom-up approach to direct the arrangement of nucleic acid nanostructures by using a supported fluid lipid bilayer as a surface scaffold. The strong attractive electrostatic interactions between RNA polyanions and cationic lipids promote RNA adsorption and self-assembly. Protocol steps for the characterization of assembled RNA complexes via several complementary methods (QCM-D, ellipsometry, confocal fluorescence microscopy, AFM) are also provided. Due to their tunable nature, lipid bilayers can be used to organize RNA laterally on the micrometer scale and thus facilitate the building of more complex 3D structures. The bilayer-based approach can be extended to other programmable RNA or DNA objects to construct intricate structures, such as 2D grids or 3D cages, with high precision.

Silver nanoclusters for RNA nanotechnology: steps towards visualization and tracking of RNA nanoparticle assemblies.

The growing interest in designing functionalized, RNA-based nanoparticles (NPs) for applications such as cancer therapeutics requires simple, efficient assembly assays. Common methods for tracking RNA assemblies such as native polyacrylamide gels and atomic force microscopy are often time-intensive and, therefore, undesirable. Here we describe a technique for rapid analysis of RNA NP assembly stages using the formation of fluorescent silver nanoclusters (Ag NCs). This method exploits the single-stranded specificity and sequence dependence of Ag NC formation to produce unique optical readouts for each stage of RNA NP assembly, obtained readily after synthesis.

Assembly of RNA nanostructures on supported lipid bilayers.

The assembly of nucleic acid nanostructures with controlled size and shape has large impact in the fields of nanotechnology, nanomedicine and synthetic biology. The directed arrangement of nano-structures at interfaces is important for many applications. In spite of this, the use of laterally mobile lipid bilayers to control RNA three-dimensional nanostructure formation on surfaces remains largely unexplored. Here, we direct the self-assembly of RNA building blocks into three-dimensional structures of RNA on fluid lipid bilayers composed of cationic 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) or mixtures of zwitterionic 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) and cationic sphingosine. We demonstrate the stepwise supramolecular assembly of discrete building blocks through specific and selective RNA-RNA interactions, based on results from quartz crystal microbalance with dissipation (QCM-D), ellipsometry, fluorescence recovery after photobleaching (FRAP) and total internal reflection fluorescence microscopy (TIRF) experiments. The assembly can be controlled to give a densely packed single layer of RNA polyhedrons at the fluid lipid bilayer surface. We show that assembly of the 3D structure can be modulated by sequence specific interactions, surface charge and changes in the salt composition and concentration. In addition, the tertiary structure of the RNA polyhedron can be controllably switched from an extended structure to one that is dense and compact. The versatile approach to building up three-dimensional structures of RNA does not require modification of the surface or the RNA molecules, and can be used as a bottom-up means of nanofabrication of functionalized bio-mimicking surfaces.

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.

Multifunctional RNA nanoparticles.

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.

RNA self-assembly and RNA nanotechnology.

CONSPECTUS: Nanotechnology's central goal involves the direct control of matter at the molecular nanometer scale to build nanofactories, nanomachines, and other devices for potential applications including electronics, alternative fuels, and medicine. In this regard, the nascent use of nucleic acids as a material to coordinate the precise arrangements of specific molecules marked an important milestone in the relatively recent history of nanotechnology. While DNA served as the pioneer building material in nucleic acid nanotechnology, RNA continues to emerge as viable alternative material with its own distinct advantages for nanoconstruction. Several complementary assembly strategies have been used to build a diverse set of RNA nanostructures having unique structural attributes and the ability to self-assemble in a highly programmable and controlled manner. Of the different strategies, the architectonics approach uniquely endeavors to understand integrated structural RNA architectures through the arrangement of their characteristic structural building blocks. Viewed through this lens, it becomes apparent that nature routinely uses thermodynamically stable, recurrent modular motifs from natural RNA molecules to generate unique and more complex programmable structures. With the design principles found in natural structures, a number of synthetic RNAs have been constructed. The synthetic nanostructures constructed to date have provided, in addition to affording essential insights into RNA design, important platforms to characterize and validate the structural self-folding and assembly properties of RNA modules or building blocks. Furthermore, RNA nanoparticles have shown great promise for applications in nanomedicine and RNA-based therapeutics. 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.

Computational and experimental characterization of RNA cubic nanoscaffolds.

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.

RNA modularity for synthetic biology.

RNA molecules are highly modular components that can be used in a variety of contexts for building new metabolic, regulatory and genetic circuits in cells. The majority of synthetic RNA systems to date predominately rely on two-dimensional modularity. However, a better understanding and integration of three-dimensional RNA modularity at structural and functional levels is critical to the development of more complex, functional bio-systems and molecular machines for synthetic biology applications.

Activation of different split functionalities on re-association of RNA-DNA hybrids.

Split-protein systems, an approach that relies on fragmentation of proteins with their further conditional re-association to form functional complexes, are increasingly used for various biomedical applications. This approach offers tight control of protein functions and improved detection sensitivity. Here we report a similar technique based on a pair of RNA-DNA hybrids that can be used generally for triggering different split functionalities. Individually, each hybrid is inactive but when two cognate hybrids re-associate, different functionalities are triggered inside mammalian cells. As a proof of concept, this work mainly focuses on the activation of RNA interference. However, the release of other functionalities (such as resonance energy transfer and RNA aptamer) is also shown. Furthermore, in vivo studies demonstrate a significant uptake of the hybrids by tumours together with specific gene silencing. This split-functionality approach presents a new route in the development of 'smart' nucleic acid-based nanoparticles and switches for various biomedical applications.

The GA-minor submotif as a case study of RNA modularity, prediction, and design.

Complex natural RNAs such as the ribosome, group I and group II introns, and RNase P exemplify the fact that three-dimensional (3D) RNA structures are highly modular and hierarchical in nature. Tertiary RNA folding typically takes advantage of a rather limited set of recurrent structural motifs that are responsible for controlling bends or stacks between adjacent helices. Herein, the GA minor and related structural motifs are presented as a case study to highlight several structural and folding principles, to gain further insight into the structural evolution of naturally occurring RNAs, as well as to assist the rational design of artificial RNAs.