Design and Characterization of Compact, Programmable, Multistranded Nonimmunostimulatory Nucleic Acid Nanoparticles Suitable for Biomedical Applications.

We report a thorough investigation of the role of single-stranded thymidine (ssT) linkers in the stability and flexibility of minimal, multistranded DNA nanostructures. We systematically explore the impact of varying the number of ssTs in three-way junction motifs (3WJs) on their formation and properties. Through various UV melting experiments and molecular dynamics simulations, we demonstrate that while the number of ssTs minimally affects thermodynamic stability, the increasing ssT regions significantly enhance the structural flexibility of 3WJs. Utilizing this knowledge, we design triangular DNA nanoparticles with varying ssTs, all showing exceptional assembly efficiency except for the 0T triangle. All triangles demonstrate enhanced stability in blood serum and are nonimmunostimulatory and nontoxic in mammalian cell lines. The 4T 3WJ is chosen as the building block for constructing other polygons due to its enhanced flexibility and favorable physicochemical characteristics, making it a versatile choice for creating cost-effective, stable, and functional DNA nanostructures that can be stored in the dehydrated forms while retaining their structures. Our study provides valuable insights into the design and application of nucleic acid nanostructures, emphasizing the importance of understanding stability and flexibility in the realm of nucleic acid nanotechnology. Our findings suggest the intricate connection between these ssTs and the structural adaptability of DNA 3WJs, paving the way for more precise design and engineering of nucleic acid nanosystems suitable for broad biomedical applications.

Context-sensitivity of isosteric substitutions of non-Watson-Crick basepairs in recurrent RNA 3D motifs.

Sequence variation in a widespread, recurrent, structured RNA 3D motif, the Sarcin/Ricin (S/R), was studied to address three related questions: First, how do the stabilities of structured RNA 3D motifs, composed of non-Watson-Crick (non-WC) basepairs, compare to WC-paired helices of similar length and sequence? Second, what are the effects on the stabilities of such motifs of isosteric and non-isosteric base substitutions in the non-WC pairs? And third, is there selection for particular base combinations in non-WC basepairs, depending on the temperature regime to which an organism adapts? A survey of large and small subunit rRNAs from organisms adapted to different temperatures revealed the presence of systematic sequence variations at many non-WC paired sites of S/R motifs. UV melting analysis and enzymatic digestion assays of oligonucleotides containing the motif suggest that more stable motifs tend to be more rigid. We further found that the base substitutions at non-Watson-Crick pairing sites can significantly affect the thermodynamic stabilities of S/R motifs and these effects are highly context specific indicating the importance of base-stacking and base-phosphate interactions on motif stability. This study highlights the significance of non-canonical base pairs and their contributions to modulating the stability and flexibility of RNA molecules.

Effective, Rapid, and Small-Scale Bioconjugation and Purification of "Clicked" Small-Molecule DNA Oligonucleotide for Nucleic Acid Nanoparticle Functionalization.

Nucleic acid-based therapeutics involves the conjugation of small molecule drugs to nucleic acid oligomers to surmount the challenge of solubility, and the inefficient delivery of these drug molecules into cells. "Click" chemistry has become popular conjugation approach due to its simplicity and high conjugation efficiency. However, the major drawback of the conjugation of oligonucleotides is the purification of the products, as traditionally used chromatography techniques are usually time-consuming and laborious, requiring copious quantities of materials. Herein, we introduce a simple and rapid purification methodology to separate the excess of unconjugated small molecules and toxic catalysts using a molecular weight cut-off (MWCO) centrifugation approach. As proof of concept, we deployed "click" chemistry to conjugate a Cy3-alkyne moiety to an azide-functionalized oligodeo-xynucleotide (ODN), as well as a coumarin azide to an alkyne-functionalized ODN. The calculated yields of the conjugated products were found to be 90.3 ± 0.4% and 86.0 ± 1.3% for the ODN-Cy3 and ODN-coumarin, respectively. Analysis of purified products by fluorescence spectroscopy and gel shift assays demonstrated a drastic amplitude of fluorescent intensity by multiple folds of the reporter molecules within DNA nanoparticles. This work is intended to demonstrate a small-scale, cost-effective, and robust approach to purifying ODN conjugates for nucleic acid nanotechnology applications.

The immunorecognition, subcellular compartmentalization, and physicochemical properties of nucleic acid nanoparticles can be controlled by composition modification.

Nucleic acid nanoparticles (NANPs) have become powerful new platforms as therapeutic and diagnostic tools due to the innate biological ability of nucleic acids to identify target molecules or silence genes involved in disease pathways. However, the clinical application of NANPs has been limited by factors such as chemical instability, inefficient intracellular delivery, and the triggering of detrimental inflammatory responses following innate immune recognition of nucleic acids. Here, we have studied the effects of altering the chemical composition of a circumscribed panel of NANPs that share the same connectivity, shape, size, charge and sequences. We show that replacing RNA strands with either DNA or chemical analogs increases the enzymatic and thermodynamic stability of NANPs. Furthermore, we have found that such composition changes affect delivery efficiency and determine subcellular localization, effects that could permit the targeted delivery of NANP-based therapeutics and diagnostics. Importantly, we have determined that altering NANP composition can dictate the degree and mechanisms by which cell immune responses are initiated. While RNA NANPs trigger both TLR7 and RIG-I mediated cytokine and interferon production, DNA NANPs stimulate minimal immune activation. Importantly, incorporation of 2'F modifications abrogates RNA NANP activation of TLR7 but permits RIG-I dependent immune responses. Furthermore, 2'F modifications of DNA NANPs significantly enhances RIG-I mediated production of both proinflammatory cytokines and interferons. Collectively this indicates that off-target effects may be reduced and/or desirable immune responses evoked based upon NANPs modifications. Together, our studies show that NANP composition provides a simple way of controlling the immunostimulatory potential, and physicochemical and delivery characteristics, of such platforms.

RNA-DNA fibers and polygons with controlled immunorecognition activate RNAi, FRET and transcriptional regulation of NF-κB in human cells.

Nucleic acid-based assemblies that interact with each other and further communicate with the cellular machinery in a controlled manner represent a new class of reconfigurable materials that can overcome limitations of traditional biochemical approaches and improve the potential therapeutic utility of nucleic acids. This notion enables the development of novel biocompatible 'smart' devices and biosensors with precisely controlled physicochemical and biological properties. We extend this novel concept by designing RNA-DNA fibers and polygons that are able to cooperate in different human cell lines and that have defined immunostimulatory properties confirmed by ex vivo experiments. The mutual intracellular interaction of constructs results in the release of a large number of different siRNAs while giving a fluorescent response and activating NF-κB decoy DNA oligonucleotides. This work expands the possibilities of nucleic acid technologies by (i) introducing very simple design principles and assembly protocols; (ii) potentially allowing for a simultaneous release of various siRNAs together with functional DNA sequences and (iii) providing controlled rates of reassociation, stabilities in human blood serum, and immunorecognition.

Retinoic acid inducible gene-I mediated detection of bacterial nucleic acids in human microglial cells.

BACKGROUND: Bacterial meningitis and meningoencephalitis are associated with devastating neuroinflammation. We and others have demonstrated the importance of glial cells in the initiation of immune responses to pathogens invading the central nervous system (CNS). These cells use a variety of pattern recognition receptors (PRRs) to identify common pathogen motifs and the cytosolic sensor retinoic acid inducible gene-1 (RIG-I) is known to serve as a viral PRR and initiator of interferon (IFN) responses. Intriguingly, recent evidence indicates that RIG-I also has an important role in the detection of bacterial nucleic acids, but such a role has not been investigated in glia. METHODS: In this study, we have assessed whether primary or immortalized human and murine glia express RIG-I either constitutively or following stimulation with bacteria or their products by immunoblot analysis. We have used capture ELISAs and immunoblot analysis to assess human microglial interferon regulatory factor 3 (IRF3) activation and IFN production elicited by bacterial nucleic acids and novel engineered nucleic acid nanoparticles. Furthermore, we have utilized a pharmacological inhibitor of RIG-I signaling and siRNA-mediated knockdown approaches to assess the relative importance of RIG-I in such responses. RESULTS: We demonstrate that RIG-I is constitutively expressed by human and murine microglia and astrocytes, and is elevated following bacterial infection in a pathogen and cell type-specific manner. Additionally, surface and cytosolic PRR ligands are also sufficient to enhance RIG-I expression. Importantly, our data demonstrate that bacterial RNA and DNA both trigger RIG-I-dependent IRF3 phosphorylation and subsequent type I IFN production in human microglia. This ability has been confirmed using our nucleic acid nanoparticles where we demonstrate that both RNA- and DNA-based nanoparticles can stimulate RIG-I-dependent IFN responses in these cells. CONCLUSIONS: The constitutive and bacteria-induced expression of RIG-I by human glia and its ability to mediate IFN responses to bacterial RNA and DNA and nucleic acid nanoparticles raises the intriguing possibility that RIG-I may be a potential target for therapeutic intervention during bacterial infections of the CNS, and that the use of engineered nucleic acid nanoparticles that engage this sensor might be a method to achieve this goal.

Functionally-interdependent shape-switching nanoparticles with controllable properties.

We introduce a new concept that utilizes cognate nucleic acid nanoparticles which are fully complementary and functionally-interdependent to each other. In the described approach, the physical interaction between sets of designed nanoparticles initiates a rapid isothermal shape change which triggers the activation of multiple functionalities and biological pathways including transcription, energy transfer, functional aptamers and RNA interference. The individual nanoparticles are not active and have controllable kinetics of re-association and fine-tunable chemical and thermodynamic stabilities. Computational algorithms were developed to accurately predict melting temperatures of nanoparticles of various compositions and trace the process of their re-association in silico. Additionally, tunable immunostimulatory properties of described nanoparticles suggest that the particles that do not induce pro-inflammatory cytokines and high levels of interferons can be used as scaffolds to carry therapeutic oligonucleotides, while particles with strong interferon and mild pro-inflammatory cytokine induction may qualify as vaccine adjuvants. The presented concept provides a simple, cost-effective and straightforward model for the development of combinatorial regulation of biological processes in nucleic acid nanotechnology.

Structure and Composition Define Immunorecognition of Nucleic Acid Nanoparticles.

Nucleic acid nanoparticles (NANPs) have evolved as a new class of therapeutics with the potential to detect and treat diseases. Despite tremendous advancements in NANP development, their immunotoxicity, one of the major impediments in clinical translation of traditional therapeutic nucleic acids (TNAs), has never been fully characterized. Here, we describe the first systematically studied immunological recognition of 25 representative RNA and DNA NANPs selected to have different design principles and physicochemical properties. We discover that, unlike traditional TNAs, NANPs used without a delivery carrier are immunoquiescent. We show that interferons (IFNs) are the key cytokines triggered by NANPs after their internalization by phagocytic cells, which agrees with predictions based on the experiences with TNAs. However, in addition to type I IFNs, type III IFNs also serve as reliable biomarkers of NANPs, which is usually not characteristic of TNAs. We show that overall immunostimulation relies on NANP shapes, connectivities, and compositions. We demonstrate that, like with traditional TNAs, plasmacytoid dendritic cells serve as the primary interferon producers among all peripheral blood mononuclear cells treated with NANPs, and scavenger receptor-mediated uptake and endosomal Toll-like receptor signaling are essential for NANP immunorecognition. The TLR involvement, however, is different from that expected for traditional TNA recognition. Based on these results, we suggest that NANP technology may serve as a prototype of auxiliary molecular language for communication with the immune system and the modulation of immune responses.

Modulating Immune Response with Nucleic Acid Nanoparticles.

Nano-objects made of nucleic acids are becoming promising materials in the biomedical field. This is, in part, due to DNA and RNA self-assembly properties that can be accurately computed to fabricate various complex nanoarchitectures of 2D and 3D shapes. The nanoparticles can be assembled from DNA, RNA, and chemically modified oligonucleotide mixtures which, in turn, influence their chemical and biophysical properties. Solid-phase synthesis allows large-scale production of individual oligonucleotide strands with batch-to-batch consistency and exceptional purity. All of these advantageous characteristics of nucleic-acid-based nanoparticles were known to be exceptionally useful as a nanoplatform for drug delivery purposes. Recently, several important discoveries have been achieved, demonstrating that nucleic acid nanoparticles (NANPs) can also be used to modulate the immune response of host cells. The purpose of this review is to briefly overview studies demonstrating architectural design principles of NANPs, as well as the ability of NANPs to control immune responses.

Programmable Nucleic Acid Based Polygons with Controlled Neuroimmunomodulatory Properties for Predictive QSAR Modeling.

In the past few years, the study of therapeutic RNA nanotechnology has expanded tremendously to encompass a large group of interdisciplinary sciences. It is now evident that rationally designed programmable RNA nanostructures offer unique advantages in addressing contemporary therapeutic challenges such as distinguishing target cell types and ameliorating disease. However, to maximize the therapeutic benefit of these nanostructures, it is essential to understand the immunostimulatory aptitude of such tools and identify potential complications. This paper presents a set of 16 nanoparticle platforms that are highly configurable. These novel nucleic acid based polygonal platforms are programmed for controllable self-assembly from RNA and/or DNA strands via canonical Watson-Crick interactions. It is demonstrated that the immunostimulatory properties of these particular designs can be tuned to elicit the desired immune response or lack thereof. To advance the current understanding of the nanoparticle properties that contribute to the observed immunomodulatory activity and establish corresponding designing principles, quantitative structure-activity relationship modeling is conducted. The results demonstrate that molecular weight, together with melting temperature and half-life, strongly predicts the observed immunomodulatory activity. This framework provides the fundamental guidelines necessary for the development of a new library of nanoparticles with predictable immunomodulatory activity.

Versatile RNA tetra-U helix linking motif as a toolkit for nucleic acid nanotechnology.

RNA nanotechnology employs synthetically modified ribonucleic acid (RNA) to engineer highly stable nanostructures in one, two, and three dimensions for medical applications. Despite the tremendous advantages in RNA nanotechnology, unmodified RNA itself is fragile and prone to enzymatic degradation. In contrast to use traditionally modified RNA strands e.g. 2'-fluorine, 2'-amine, 2'-methyl, we studied the effect of RNA/DNA hybrid approach utilizing a computer-assisted RNA tetra-uracil (tetra-U) motif as a toolkit to address questions related to assembly efficiency, versatility, stability, and the production costs of hybrid RNA/DNA nanoparticles. The tetra-U RNA motif was implemented to construct four functional triangles using RNA, DNA and RNA/DNA mixtures, resulting in fine-tunable enzymatic and thermodynamic stabilities, immunostimulatory activity and RNAi capability. Moreover, the tetra-U toolkit has great potential in the fabrication of rectangular, pentagonal, and hexagonal NPs, representing the power of simplicity of RNA/DNA approach for RNA nanotechnology and nanomedicine community.

Programmable folding of fusion RNA in vivo and in vitro driven by pRNA 3WJ motif of phi29 DNA packaging motor.

Misfolding and associated loss of function are common problems in constructing fusion RNA complexes due to changes in energy landscape and the nearest-neighbor principle. Here we report the incorporation and application of the pRNA-3WJ motif of the phi29 DNA packaging motor into fusion RNA with controllable and predictable folding. The motif included three discontinuous ∼18 nucleotide (nt) fragments, displayed a distinct low folding energy (Shu D et al., Nature Nanotechnology, 2011, 6:658-667), and folded spontaneously into a leading core that enabled the correct folding of other functionalities fused to the RNA complex. Three individual fragments dispersed at any location within the sequence allowed the other RNA functional modules to fold into their original structures with authentic functions, as tested by Hepatitis B virus ribozyme, siRNA, and aptamers for malachite green (MG), spinach, and streptavidin (STV). Only nine complementary nucleotides were present for any two of the three ∼18-nt fragments, but the three 9 bp branches were so powerful that they disrupted other double strands with more than 15 bp within the fusion RNA. This system enabled the production of fusion complexes harboring multiple RNA functionalities with correct folding for potential applications in biotechnology, nanomedicine and nanotechnology. We also applied this system to investigate the principles governing the folding of RNA in vivo and in vitro. Temporal production of RNA sequences during in vivo transcription caused RNA to fold into different conformations that could not be predicted with routine principles derived from in vitro studies.

Enhancing immunomodulation on innate immunity by shape transition among RNA triangle, square and pentagon nanovehicles.

Modulation of immune response is important in cancer immunotherapy, vaccine adjuvant development and inflammatory or immune disease therapy. Here we report the development of new immunomodulators via control of shape transition among RNA triangle, square and pentagon. Changing one RNA strand in polygons automatically induced the stretching of the interior angle from 60° to 90° or 108°, resulting in self-assembly of elegant RNA triangles, squares and pentagons. When immunological adjuvants were incorporated, their immunomodulation effect for cytokine TNF-α and IL-6 induction was greatly enhanced in vitro and in animals up to 100-fold, while RNA polygon controls induced unnoticeable effect. The RNA nanoparticles were delivered to macrophages specifically. The degree of immunostimulation greatly depended on the size, shape and number of the payload per nanoparticles. Stronger immune response was observed when the number of adjuvants per polygon was increased, demonstrating the advantage of shape transition from triangle to pentagon.

Entropy-driven one-step formation of Phi29 pRNA 3WJ from three RNA fragments.

The emerging field of RNA nanotechnology necessitates creation of functional RNA nanoparticles but has been limited by particle instability. It has been shown that the three-way junction of bacteriophage phi29 motor pRNA has unusual stability and can self-assemble from three fragments with high efficiency. It is generally believed that RNA and DNA folding is energy landscape-dependent, and the folding of RNA is driven by enthalpy. Here we examine the thermodynamic characteristics of the 3WJ components as 2'-fluoro RNA, DNA, and RNA. It was seen that the three fragments existed either in 3WJ complex or as monomers, with the intermediate of dimers almost undetectable. It seems that the three fragments can lead to the formation of the 3WJ complex efficiently within a rapid time. A low dissociation constant (apparent KD) of 11.4 nM was determined for RNA, inclusion of 2'-F pyrimidines strengthened the KD to 4.5 nM, and substitution of DNA weakened it to 47.7 nM. The ΔG°37, were -36, -28, and -15 kcal/mol for 3WJ2'-F, 3WJRNA, and 3WJDNA, respectively. It is found that the formation of the three-component complex was governed by entropy, instead of enthalpy, as usually found in RNA complexes. Here entropy-driven is referring to a dominating entropic contribution to the increased stability of the 3WJ(2'-F and 3WJ(RNA) compared to the 3WJ(DNA,) instead of referring to the absolute role or total energy governing 3WJ folding. [corrected].

Bioconjugation of Functionalized Oligodeoxynucleotides with Fluorescence Reporters for Nanoparticle Assembly.

In the field of nucleic acid nanotechnology and therapeutics, there is an imperative need to improve the oligodeoxynucleotides' (ODNs) properties by either chemical modification of the oligonucleotides' structure or to covalently link them to a reporter or therapeutic moieties that possess biologically relevant properties. The chemical conjugation can thus significantly improve the intrinsic properties not only of ODNs but also reporter/therapeutic molecules. Bioconjugation of nucleic acids to small molecules also serves as a nano-delivery facility to transport various functionalities to specific targets. Herein, we describe a generalized methodology that deploys azide-alkyne cycloaddition, a click reaction to conjugate a cyanine-3 alkyne moiety to an azide-functionalized ODN 12-mer, as well as 3-azido 7-hydroxycoumarin to an alkyne functionalized ODN 12-mer.

Thermodynamic Characterization of Nucleic Acid Nanoparticles Hybridization by UV Melting.

The advances in nucleic acid nanotechnology have given rise to various elegantly designed structural complexes fabricated from DNA, RNA, chemically modified RNA strands, and their mixtures. The structural properties of NA nanoparticles (NANP) generally dictate and significantly impact biological function; and thus, it is critical to extract information regarding relative stabilities of the different structural forms. The adequate stability assessment requires knowledge of thermodynamic parameters that can be empirically derived using conventional UV-melting technique. The focus of this chapter is to describe methodology to evaluate thermodynamic data of NANPs complexation based on DNA 12 base-pair (bp) duplex formation as an example.

Toehold-Mediated Shape Transition of Nucleic Acid Nanoparticles.

We introduce a toehold-mediated strand displacement strategy for regulated shape-switching of nucleic acid nanoparticles (NANPs) enabling their sequential transformation from triangular to hexagonal architectures at isothermal conditions. The successful shape transitions were confirmed by electrophoretic mobility shift assays, atomic force microscopy, and dynamic light scattering. Furthermore, implementation of split fluorogenic aptamers allowed for monitoring the individual transitions in real time. Three distinct RNA aptamers─malachite green (MG), broccoli, and mango─were embedded within NANPs as reporter domains to confirm shape transitions. While MG "lights up" within the square, pentagonal, and hexagonal constructs, the broccoli is activated only upon formation of pentagon and hexagon NANPs, and mango reports only the presence of hexagons. Moreover, the designed RNA fluorogenic platform can be employed to construct a logic gate that performs an AND operation with three single-stranded RNA inputs by implementing a non-sequential polygon transformation approach. Importantly, the polygonal scaffolds displayed promising potential as drug delivery agents and biosensors. All polygons exhibited effective cellular internalization followed by specific gene silencing when decorated with fluorophores and RNAi inducers. This work offers a new perspective for the design of toehold-mediated shape-switching nanodevices to activate different light-up aptamers for the development of biosensors, logic gates, and therapeutic devices in the nucleic acid nanotechnology.

Fluorogenic RNA Aptamers: A Nano-platform for Fabrication of Simple and Combinatorial Logic Gates.

RNA aptamers that bind non-fluorescent dyes and activate their fluorescence are highly sensitive, nonperturbing, and convenient probes in the field of synthetic biology. These RNA molecules, referred to as light-up aptamers, operate as molecular nanoswitches that alter folding and fluorescence function in response to ligand binding, which is important in biosensing and molecular computing. Herein, we demonstrate a conceptually new generation of smart RNA nano-devices based on malachite green (MG)-binding RNA aptamer, which fluorescence output controlled by addition of short DNA oligonucleotides inputs. Four types of RNA switches possessing AND, OR, NAND, and NOR Boolean logic functions were created in modular form, allowing MG dye binding affinity to be changed by altering 3D conformation of the RNA aptamer. It is essential to develop higher-level logic circuits for the production of multi-task nanodevices for data processing, typically requiring combinatorial logic gates. Therefore, we further designed and synthetized higher-level half adder logic circuit by "in parallel" integration of two logic gates XOR and AND within a single RNA nanoparticle. The design utilizes fluorescence emissions from two different RNA aptamers: MG-binding RNA aptamer (AND gate) and Broccoli RNA aptamer that binds DFHBI dye (XOR gate). All computationally designed RNA devices were synthesized and experimentally tested in vitro. The ability to design smart nanodevices based on RNA binding aptamers offers a new route to engineer "label-free" ligand-sensing regulatory circuits, nucleic acid detection systems, and gene control elements.

Evaluation of Thermal Stability of RNA Nanoparticles by Temperature Gradient Gel Electrophoresis (TGGE) in Native Condition.

Temperature gradient gel electrophoresis (TGGE) is a powerful tool used to analyze the thermal stabilities of nucleic acids. While TGGE is a decades-old technique, it has recently gained favor in the field of RNA nanotechnology, notably in assessing the thermal stabilities of RNA nanoparticles (NPs). With TGGE, an electrical current and a linear temperature gradient are applied simultaneously to NP-loaded polyacrylamide gel, separating the negatively charged NPs based on their thermal behavior (a more stable RNA complex will remain intact through higher temperature ranges). The linear temperature gradient can be set either perpendicular or parallel to the electrical current, as either will make the NPs undergo a transition from native to denatured conformations. Often, the melting transition is influenced by sequence variations, secondary/tertiary structures, concentrations, and external factors such as the presence of a denaturing agent (e.g., urea), the presence of monovalent or divalent metal ions, and the pH of the solvent. In this chapter, we describe the experimental setup and the analysis of the thermal stability of RNA NPs in native conditions using a modified version of a commercially available TGGE system.

Fabrication of RNA 3D Nanoprisms for Loading and Protection of Small RNAs and Model Drugs.

Constructing containers with defined shape and size to load and protect therapeutics and subsequently control their release in the human body has long been a dream. The fabrication of 3D RNA prisms, characterized by atomic force microscopy, cryo-electron microscopy, dynamic light scattering, and polyacrylamide gel electrophoresis, is reported for the loading and protection of small molecules, proteins, small RNA molecules, and their controlled release.