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

An information-bearing seed for nucleating algorithmic self-assembly

Self-assembly creates natural mineral, chemical, and biological structures of great complexity. Often, the same starting materials have the potential to form an infinite variety of distinct structures; information in a seed molecule can determine which form is grown as well as where and when. These phenomena can be exploited to program the growth of complex supramolecular structures, as demonstrated by the algorithmic self-assembly of DNA tiles. However, the lack of effective seeds has limited the reliability and yield of algorithmic crystals. Here, we present a programmable DNA origami seed that can display up to 32 distinct binding sites and demonstrate the use of seeds to nucleate three types of algorithmic crystals. In the simplest case, the starting materials are a set of tiles that can form crystalline ribbons of any width; the seed directs assembly of a chosen width with >90% yield. Increased structural diversity is obtained by using tiles that copy a binary string from layer to layer; the seed specifies the initial string and triggers growth under near-optimal conditions where the bit copying error rate is 17 kb of sequence information. In sum, this work demonstrates how DNA origami seeds enable the easy, high-yield, low-error-rate growth of algorithmic crystals as a route toward programmable bottom-up fabrication

Multicomponent Conjugates of Anticancer Drugs and Monoclonal Antibody with PAMAM Dendrimers to Increase Efficacy of HER-2 Positive Breast Cancer Therapy

Purpose: Conjugation of nanocarriers with antibodies that bind to specific membrane receptors that are overexpressed in cancer cells enables targeted delivery. In the present study, we developed and synthesised two PAMAM dendrimer-trastuzumab conjugates that carried docetaxel or paclitaxel, specifically targeted to cells which overexpressed HER-2. Methods: The 1H NMR, 13C NMR, FTIR and RP-HPLC were used to analyse the characteristics of the products and assess their purity. The toxicity of PAMAM-trastuzumab, PAMAM-doc-trastuzumab and PAMAM-ptx-trastuzumab conjugates was determined using MTT assay and compared with free trastuzumab, docetaxel and paclitaxel toward HER-2-positive (SKBR-3) and negative (MCF-7) human breast cancer cell lines. The cellular uptake and internal localisation were studied using flow cytometry and confocal microscopy, respectively. Results: The PAMAM-drug-trastuzumab conjugates in particular showed extremely high toxicity toward the HER-2-positive SKBR-3 cells and very low toxicity towards to HER-2-negative MCF-7 cells. As expected, the HER-2-positive SKBR-3 cell line accumulated trastuzumab from both conjugates rapidly; but surprisingly, although a large amount of PAMAM-ptx-trastuzumab conjugate was observed in the HER-2-negative MCF-7 cells. Confocal microscopy confirmed the intracellular localisation of analysed compounds. The key result of fluorescent imaging was the identification of strong selective binding of the PAMAM-doc-trastuzumab conjugate with HER-2-positive SKBR-3 cells only. Conclusions: Our results confirm the high selectivity of PAMAM-doc-trastuzumab and PAMAM-ptx-trastuzumab conjugates for HER-2-positive cells, and demonstrate the utility of trastuzumab as a targeting agent. Therefore, the analysed conjugates present an promising approach for the improvement of efficacy of targeted delivery of anticancer drugs such as docetaxel or paclitaxel. © 2019, The Author(s)

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

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

Engineering cooperative tecto–RNA complexes having programmable stoichiometries

High affinity and specificity RNA–RNA binding interfaces can be constructed by combining pairs of GNRA loop/loop–receptor interaction motifs. These interactions can be fused using flexible four-way junction motifs to create divalent, self-assembling scaffolding units (‘tecto-RNA’) that have favorable properties for nanomedicine and other applications. We describe the design and directed assembly of tecto-RNA units ranging from closed, cooperatively assembling ring-shaped complexes of programmable stoichiometries (dimers, trimers and tetramers) to open multimeric structures. The novelty of this work is that tuning of the stoichiometries of self-assembled complexes is achieved by precise positioning of the interaction motifs in the monomer units rather than changing their binding specificities. Structure-probing and transmission electron microscopy studies as well as thermodynamic analysis support formation of closed cooperative complexes that are highly resistant to nuclease digestion. The present designs provide two helical arms per RNA monomer for further functionalization aims

The Dimeric Proto-Ribosome: Structural Details and Possible Implications on the Origin of Life

A symmetric pocket-like entity, composed of two L-shaped RNA units, encircles the peptide synthesis site within the contemporary ribosome. This entity was suggested to be the vestige of a dimeric proto-ribosome, which could have formed spontaneously in the prebiotic world, catalyzing non-coded peptide bond formation and elongation. This structural element, beyond offering the initial step in the evolution of translation, is hypothesized here to be linked to the origin of life. By catalyzing the production of random peptide chains, the proto-ribosome could have enabled the formation of primary enzymes, launching a process of co-evolution of the translation apparatus and the proteins, thus presenting an alternative to the RNA world hypothesis

Oxidised guanidinohydantoin (Ghox) and spiroiminodihydantoin (Sp) are major products of iron- and copper-mediated 8-oxo-7,8-dihydroguanine and 8-oxo-7,8-dihydro-2-deoxyguanosine oxidation

8-Oxo-7,8-dihydroguanine (8-oxoGua), an important biomarker of DNA damage in oxidatively generated stress, is highly reactive towards further oxidation. Much work has been carried out to investigate the oxidation products of 8-oxoGua by one-electron oxidants, singlet oxygen, and peroxynitrite. This report details for the first time, the iron- and copper-mediated Fenton oxidation of 8-oxoGua and 8-oxo-7,8-dihydro-29-deoxyguanosine (8-oxodGuo). Oxidised guanidinohydantoin (Ghox) was detected as the major product of oxidation of 8-oxoGua with iron or copper and hydrogen peroxide, both at pH 7 and pH 11. Oxaluric acid was identified as a final product of 8-oxoGua oxidation. 8-oxodGuo was subjected to oxidation under the same conditions as 8-oxoGua. However, dGhox was not generated. Instead, spiroiminodihydantoin (Sp) was detected as the major product for both iron and copper mediated oxidation at pH 7. It was proposed that the oxidation of 8-oxoGua was initiated by its one-electron oxidation by the metal species, which leads to the reactive intermediate 8-oxoGua?+, which readily undergoes further oxidation. The product of 8-oxoGua and 8-oxodGuo oxidation was determined by the 29-deoxyribose moiety of the 8-oxodGuo, not whether copper or iron was the metal involved in the oxidation

Three critical hydrogen bonds determine the catalytic activity of the Diels–Alderase ribozyme

Compared to protein enzymes, our knowledge about how RNA accelerates chemical reactions is rather limited. The crystal structures of a ribozyme that catalyzes Diels–Alder reactions suggest a rich tertiary architecture responsible for catalysis. In this study, we systematically probe the relevance of crystallographically observed ground-state interactions for catalytic function using atomic mutagenesis in combination with various analytical techniques. The largest energetic contribution apparently arises from the precise shape complementarity between transition state and catalytic pocket: A single point mutant that folds correctly into the tertiary structure but lacks one H-bond that normally stabilizes the pocket is completely inactive. In the rate-limiting chemical step, the dienophile is furthermore activated by two weak H-bonds that contribute ∼7–8 kJ/mol to transition state stabilization, as indicated by the 25-fold slower reaction rates of deletion mutants. These H-bonds are also responsible for the tight binding of the Diels–Alder product by the ribozyme that causes product inhibition. For high catalytic activity, the ribozyme requires a fine-tuned balance between rigidity and flexibility that is determined by the combined action of one inter-strand H-bond and one magnesium ion. A sharp 360° turn reminiscent of the T-loop motif observed in tRNA is found to be important for catalytic function