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

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

Gold Nanoparticles in Conjunction with Nucleic Acids as a Modern Molecular System for Cellular Delivery

Development of nanotechnology has become prominent in many fields, such as medicine, electronics, production of materials, and modern drugs. Nanomaterials and nanoparticles have gained recognition owing to the unique biochemical and physical properties. Considering cellular application, it is speculated that nanoparticles can transfer through cell membranes following different routes exclusively owing to their size (up to 100 nm) and surface functionalities. Nanoparticles have capacity to enter cells by themselves but also to carry other molecules through the lipid bilayer. This quality has been utilized in cellular delivery of substances like small chemical drugs or nucleic acids. Different nanoparticles including lipids, silica, and metal nanoparticles have been exploited in conjugation with nucleic acids. However, the noble metal nanoparticles create an alternative, out of which gold nanoparticles (AuNP) are the most common. The hybrids of DNA or RNA and metal nanoparticles can be employed for functional assemblies for variety of applications in medicine, diagnostics or nano-electronics by means of biomarkers, specific imaging probes, or gene expression regulatory function. In this review, we focus on the conjugates of gold nanoparticles and nucleic acids in the view of their potential application for cellular delivery and biomedicine. This review covers the current advances in the nanotechnology of DNA and RNA-AuNP conjugates and their potential applications. We emphasize the crucial role of metal nanoparticles in the nanotechnology of nucleic acids and explore the role of such conjugates in the biological systems. Finally, mechanisms guiding the process of cellular intake, essential for delivery of modern therapeutics, will be discussed

Tissue-Nonspecific Alkaline Phosphatase (TNAP) as the Enzyme Involved in the Degradation of Nucleotide Analogues in the Ligand Docking and Molecular Dynamics Approaches

Tissue-nonspecific alkaline phosphatase (TNAP) is known to be involved in the degradation of extracellular ATP via the hydrolysis of pyrophosphate (PPi). We investigated, using three different computational methods, namely molecular docking, thermodynamic integration (TI) and conventional molecular dynamics (MD), whether TNAP may also be involved in the utilization of β,γ-modified ATP analogues. For that, we analyzed the interaction of bisphosphonates with this enzyme and evaluated the obtained structures using in silico studies. Complexes formed between pyrophosphate, hypophosphate, imidodiphosphate, methylenediphosphonic acid monothiopyrophosphate, alendronate, pamidronate and zoledronate with TNAP were generated and analyzed based on ligand docking, molecular dynamics and thermodynamic integration. The obtained results indicate that all selected ligands show high affinity toward this enzyme. The forming complexes are stabilized through hydrogen bonds, electrostatic interactions and van der Waals forces. Short- and middle-term molecular dynamics simulations yielded very similar affinity results and confirmed the stability of the protein and its complexes. The results suggest that certain effectors may have a significant impact on the enzyme, changing its properties

Highly Fluorescent Distyrylnaphthalene Derivatives as a Tool for Visualization of Cellular Membranes

Fluorescent imaging, which is an important interdisciplinary field bridging research from organic chemistry, biochemistry and cell biology has been applied for multi-dimensional detection, visualization and characterization of biological structures and processes. Especially valuable is the possibility to monitor cellular processes in real time using fluorescent probes. In this work, conjugated oligoelectrolytes and neutral derivatives with the distyrylnaphthalene core (SN-COEs) were designed, synthetized and tested for biological properties as membrane-specific fluorescent dyes for the visualization of membrane-dependent cellular processes. The group of tested compounds includes newly synthesized distyrylnaphthalene derivatives (DSNNs): a trimethylammonium derivative (DSNN-NMe3+), a phosphonate derivative (DSNN-P), a morpholine derivative (DSNN-Mor), a dihydroxyethylamine derivative (DSNN-DEA), a phosphonate potassium salt (DSNN-POK), an amino derivative (DSNN-NH2) and pyridinium derivative (DSNN-Py+). All compounds were tested for their biological properties, including cytotoxicity and staining efficiency towards mammalian cells. The fluorescence intensity of SN-COEs incorporated into cellular structures was analyzed by fluorescence activated cell sorting (FACS) and photoluminescence spectroscopy. The cytotoxicity results have shown that all tested SN-COEs can be safely used in the human and animal cell studies. Fluorescence and confocal microscopy observations confirm that tested COEs can be applied as fluorescent probes for the visualization of intracellular membrane components in a wide range of different cell types, including adherent and suspension cells. The staining procedure may be performed under both serum free and complete medium conditions. The presented studies have revealed the interesting biological properties of SN-COEs and confirmed their applicability as dyes for staining the membranous structures of eukaryotic cells, which may be useful for visualization of wide range of biological processes dependent of the extra-/intracellular communications and/or based on the remodeling of cellular membranes