Controlling RNA self-assembly to form filaments

Fundamental control over supra-molecular self-assembly for organization of matter on the nano-scale is a major objective of nanoscience and nanotechnology. ‘RNA tectonics’ is the design of modular RNA units, called tectoRNAs, that can be programmed to self-assemble into novel nano- and meso-scopic architectures of desired size and shape. We report the three-dimensional design of tectoRNAs incorporating modular 4-way junction (4WJ) motifs, hairpin loops and their cognate loop–receptors to create extended, programmable interaction interfaces. Specific and directional RNA–RNA interactions at these interfaces enable conformational, topological and orientational control of tectoRNA self-assembly. The interacting motifs are precisely positioned within the helical arms of the 4WJ to program assembly from only one helical stacking conformation of the 4WJ. TectoRNAs programmed to assemble with orientational compensation produce micrometer-scale RNA filaments through supra-molecular equilibrium polymerization. As visualized by transmission electron microscopy, these RNA filaments resemble actin filaments from the protein world. This work emphasizes the potential of RNA as a scaffold for designing and engineering new controllable biomaterials mimicking modern cytoskeletal proteins

The architectonics of programmable RNA and DNA nanostructures

The past several years have witnessed the emergence of a new world of nucleic-acid-based architectures with highly predictable and programmable self-assembly properties. For almost two decades, DNA has been the primary material for nucleic acid nanoconstruction. More recently, the dramatic increase in RNA structural information led to the development of RNA architectonics, the scientific study of the principles of RNA architecture with the aim of constructing RNA nanostructures of any arbitrary size and shape. The remarkable modularity and the distinct but complementary nature of RNA and DNA nanomaterials are revealed by the various selfassembly strategies that aim to achieve control of the arrangement of matter at a nanoscale level. Introduction The complex supramolecular (see glossary) structures that emerged in living organisms through billions of years of evolution rely on two basic self-assembly processes: the spontaneous folding of one polymer chain into a stable well-defined 3D structure; and the assembly of multiple subunits into defined, modular supramolecular architectures. Key characteristics are hierarchical organization, modular components, and stereochemically specific and selective interactions. Programmable assembly (see glossary) results from the application of folding and assembly principles gleaned from biological structures to design molecules that will, in a predictable manner, fold into specific shapes and subsequently assemble with one another into supramolecular architectures according to the structural information encoded within their primary structure. Although programmable self-assembly is at the core of supramolecular chemistry Proteins are the material of choice for building the structural, catalytic and regulatory components of cells, but their folding and assembly remain challenging to predict and design because of the inherent complexity of their 3D structures (see the reviews from Ranganathan, Waters, Kuhlman and Chin in this issue). By contrast, DNA, as the carrier of the genetic information in cells, has only four deoxynucleotide chemical building blocks, a high chemical stability, and predictable folding and assembly properties that are readily amenable to the rational design and construction of 3D nanostructures by programmable self-assembly [2,3,4 -6 ]. RNA has recently emerged as a challenger to DNA, interesting in its own right as a medium for programmable nanoconstruction (e.g. [7,8,9 ,10,11 ,12 ]). Despite a chemical structure very similar to that of DNA, RNA is chemically more labile than DNA, but is also more prone to fold into complex tertiary structures with recognition and catalytic properties reminiscent of those of proteins. Natural RNAs are the working components of biologically important molecular machines that are capable of using cellular energy in the form of ATP or GTP to perform mechanical work and to carry out complex tasks of information processing, such as template-directed protein synthesis and multiplexed gene regulation Basic structural properties and modularity of RNA and DNA nanostructures RNA and DNA modularity is hierarchically expressed at a chemical, structural and supramolecular level Despite a limited number of known DNA tertiary (38) structure motifs (see glossary; At a 48 structure level, RNA and DNA modular units assemble further into complex and highly modular supramolecular architectures in a predictable manner using base-pair rules as organizational instructions. The dimensionality of these nanostructures is directly related to the number, shape, geometry and orientation of cohesive, assembling interfaces formed between constitutive RNA or DNA tiles (see glossary) [6 ] ( DNA architectonics: variations on the same structural theme Because of the lack of stable natural 38 structure motifs, much effort has been expended designing robust and rigid DNA self-assembling building blocks A subtle balance of flexibility and stress is required for building good self-assembling tiles The monolithic structure of most DNA tiles imposes strong geometrical constraints on the positioning of their cohesive interfaces In the future, the use of triple helices RNA architectonics: sculpting new RNA structures The concept of RNA tectonics (see glossary) was initially defined as referring to the modular character of RNA structures that can be decomposed and reassembled to create new modular RNA units, called tectoRNAs (see glossary), which are able to self-assemble into nanoscale and mesoscale architectures of any desired size and shape b The persistence lengths of RNA and DNA were determined experimentally by single-molecule analysis (e.g. Current Opinion in Structural Biology The characterization of tectoRNA folding and self-assembly properties is typically performed by biochemical and biophysical methods, and visualization techniques, such as atomic force microscopy (AFM) [9 ,47] and transmission electron microscopy (TEM) Although still a new field of investigation, RNA architectonics has already generated a great variety of tectoRNA units able to assemble into highly modular supramolecular architectures of arbitrary shapes ( Nanoparticles, filaments and 2D RNA architectures The first tectoRNAs to be generated by RNA architectonics self-assemble through loop-receptor interfaces to form dimeric nanoparticles Collinear kissing loop interactions can generate strong 48 intermolecular interfaces to promote the formation of RNA particles of different sizes [50] The high modularity and hierarchical supramolecular structure of tectosquares makes it possible to construct a large number of them from a limited set of tectoRNAs that assemble through strong 48 interaction loop-loop interfaces Strategies for programmable nucleic acid self-assembly Two main approaches can be distinguished for programmable self-assembly of nucleic acid architectures ( Figure 4 Programmable supramolecular RNA architectures. (a) 0D loop-receptor (RL) dimeric tectoRNA particle: the original 38 structure model (left) Stepwise assembly can be used to generate programmable architectures of finite size, with the position of each of the constitutive molecules known and therefore addressable within the final architecture. The first demonstration of this approach led to the fabrication of RNA nanogrids of finite size Each of these approaches can make use of additional nonmutually exclusive self-assembly strategies, such as algorithmic self-assembly, directed nucleation (or templated) self-assembly and scaffolded self-assembly. In algorithmic self-assembly, a set of nucleic acid tiles, defined as Wang tiles (see glossary), is viewed as the algorithm for a particular computational task leading to the formation of 1D, 2D and 3D patterns. This strategy was used to compute the formation of aperiodic fractal 2D patterns based on the Sierpinski triangle pattern Nucleic acid architectonics Jaeger and Chworos 539 The main strategies for programmable self-assembly. (a) Single-step self-assembly: all the molecules are mixed together and assembled through a slow cool annealing procedure (most DNA architectures are formed this way). (b) Stepwise hierarchical self-assembly [9 ,58]: specific sets of molecules are first separately assembled into small supramolecular entities that are then mixed in a stepwise fashion to form the final architecture. Hierarchical assembly is favored by the use of 48 interactions with different stabilities and magnesium requirements. (c) Scaffolded self-assembly or scaffolded DNA origami: a long singlestranded molecule is folded into an arbitrary shape with small oligonucleotides acting as staples Additional principles of nucleic acid architectonics Principle of orientational compensation The inherent asymmetric nature of RNA and DNA tiles can have a dramatic effect on the larger nanostructures that they form by introducing various degrees of curvature. By using the principle of orientational compensation, whereby two adjacent units are related by a local twofold pseudo-rotational axis of symmetry, one source of asymmetry can be locally eliminated, so that asymmetric tiles that are not perfectly flat can still assemble in a plane instead of forming nanotube

Tubulopathy in nephrolithiasis: Consequence rather than cause

Tubulopathy in nephrolithiasis: Consequence rather than cause. To address whether a renal tubular dysfunction is encountered in a particular patient subgroup with urolithiasis, the following parameters of tubular function were measured in urine taken in the morning from 214 stone formers after fasting: pH, excretion of lysozyme and γ-glutamyl transferase (γ-GT); fractional excretion (FE) of glucose, insulin, Mg, K, and HCO3 after an alkali loading; and the renal threshold for phosphate (TmP/GFR). The following diagnoses were made in the patient group: primary hyperparathyroidism (N = 8), medullary sponge kidneys (N = 21), hyperuricemia (N = 10), cystinuria (N = 2), struvite stone disease (N = 6), idiopathic hypercalciuria of the absorptive (N = 25), dietary (N = 69) or renal (N = 7) type, and normocalciuric idiopathic urolithiasis (N = 66). In 31% of the patients TmP/GFR was below 0.80 mmole/liter and in 13% of the patients, FE HCO3 after alkali loading was above normal. Urinary excretion of lysozyme and that of γ-GT both were elevated in 17% of the patients. FE glucose, FE insulin, FE Mg, and FE K were elevated in 8, 9, 3, and 7% of the patients, respectively. This study demonstrates that a significant number of stone formers present with signs of renal tubular dysfunction, primarily involving the proximal tubule since apparent leaks of phosphate and of bicarbonate were most frequently encountered. The defects were not specific for a given etiologic group of patients; on the other hand, occurrence was related to the presence of large stones in the pyelocaliceal system at the time data were gathered. Taken together these data suggest that the tubulopathy in nephrolithiasis is the consequence rather than the cause of the stone

Promoting RNA helical stacking via A-minor junctions

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

Attenuation of loop-receptor interactions with pseudoknot formation

RNA tetraloops can recognize receptors to mediate long-range interactions in stable natural RNAs. In vitro selected GNRA tetraloop/receptor interactions are usually more ‘G/C-rich’ than their ‘A/U-rich’ natural counterparts. They are not as widespread in nature despite comparable biophysical and chemical properties. Moreover, while AA, AC and GU dinucleotide platforms occur in natural GAAA/11 nt receptors, the AA platform is somewhat preferred to the others. The apparent preference for ‘A/U-rich’ GNRA/receptor interactions in nature might stem from an evolutionary adaptation to avoid folding traps at the level of the larger molecular context. To provide evidences in favor of this hypothesis, several riboswitches based on natural and artificial GNRA receptors were investigated in vitro for their ability to prevent inter-molecular GNRA/receptor interactions by trapping the receptor sequence into an alternative intra-molecular pseudoknot. Extent of attenuation determined by native gel-shift assays and co-transcriptional assembly is correlated to the G/C content of the GNRA receptor. Our results shed light on the structural evolution of natural long-range interactions and provide design principles for RNA-based attenuator devices to be used in synthetic biology and RNA nanobiotechnology

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

Comprehensive features of natural and in vitro selected GNRA tetraloop-binding receptors

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

The UA_handle: a versatile submotif in stable RNA architectures†

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

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