Nucleic Acid Nanotechnology Research Articles

A Catalytic Hairpin Assembly-Based Approach for RNA Imaging in Living Cells.

The advancement of nucleic acid nanotechnology has resulted in broad applications of DNA- and RNA-based molecular sensors for bioanalysis. Catalytic hairpin assembly is such a type of programmable and enzyme-free nucleic acid circuit that has been popularly used in developing biosensors. Genetically encodable fluorogenic RNA-based devices have recently gained a lot of attentions as a powerful tool for intracellular imaging. Combining these two techniques together, we have developed a genetically encodable RNA-based catalytic hairpin assembly circuit for the highly sensitive detection of low-abundance RNAs in living cells. In this system, the binding of one target RNA can catalytically trigger the generation of tens-to-hundreds of fluorogenic RNA reporters and produce a significantly enhanced fluorescence signal. Here, we will introduce the detailed design, optimization, and imaging protocol of this new type of powerful imaging tools.

Six-Letter DNA Nanotechnology: Incorporation of Z-P Base Pairs into Self-Assembling 3D Crystals.

Artificially expanded genetic information systems (AEGIS) were developed to expand the diversity and functionality of biological systems. Recent experiments have shown that these expanded DNA molecular systems are robust platforms for information storage and retrieval as well as useful for basic biotechnologies. In tandem, nucleic acid nanotechnology has seen the use of information-based "semantomorphic" encoding to drive the self-assembly of a vast array of supramolecular devices. To establish the effectiveness of AEGIS toward nanotechnological applications, we investigated the ability of a six-letter alphabet composed of A:T, G:C and synthetic Z:P (Z, 6-amino-3-(1'-β-d-2'-deoxy ribofuranosyl)-5-nitro-(1H)-pyridin-2-one; P, 2-amino-8-(1'-β-d-2'-deoxyribofuranosyl)-imidazo-[1,2a]-1,3,5-triazin-(8H)-4-one) base pairs to engage in 3D self-assembly. We found that crystals could be programmably assembled from AEGIS oligomers. We conclude that unnatural base pairs can be used for the topological self-assembly of crystals. We anticipate the expansion of AEGIS-based nucleic acid nanotechnologies to enable the development of novel nanomaterials, high-fidelity signal cascades, and dynamic nanoscale devices.

In Vivo Interactions of Nucleic Acid Nanostructures With Cells.

Nucleic acid nanostructures, derived from the assembly of nucleic acid building blocks (e.g., plasmids and oligonucleotides), are important intracellular carriers of therapeutic cargoes widely utilized in preclinical nanomedicine applications, yet their clinical translation remains scarce. In the era of "translational nucleic acid nanotechnology", a deeper mechanistic understanding of the interactions of nucleic acid nanostructures with cells in vivo will guide the development of more efficacious nanomedicines. This review showcases the recent progress in dissecting the in vivo interactions of four key types of nucleic acid nanostructures (i.e., tile-based, origami, spherical nucleic acid, and nucleic acid nanogel) with cells in rodents over the past five years. Emphasis lies on the cellular-level distribution of nucleic acid nanostructures in various organs and tissues and the cellular responses induced by their cellular entry. Next, in the spirit of preclinical translation, this review features the latest interactions of nucleic acid nanostructures with cells in large animals and humans. Finally, the review offers directions for studying the interactions of nucleic acid nanostructures with cells from both materials and biology perspectives and concludes with some regulatory updates.

Single-molecule force spectroscopy of toehold-mediated strand displacement

Toehold-mediated strand displacement (TMSD) is extensively utilized in dynamic DNA nanotechnology and for a wide range of DNA or RNA-based reaction circuits. Investigation of TMSD kinetics typically relies on bulk fluorescence measurements providing effective, bulk-averaged reaction rates. Information on individual molecules or even base pairs is scarce. In this work, we explore the dynamics of strand displacement processes at the single-molecule level using single-molecule force spectroscopy with a microfluidics-enhanced optical trap supported by state-of-the-art coarse-grained simulations. By applying force, we can trigger and observe TMSD in real-time with microsecond and nanometer resolution. We find TMSD proceeds very rapidly under load with single step times of 1 µs. Tuning invasion efficiency by introducing mismatches allows studying thousands of forward/backward invasion events on a single molecule and analyze the kinetics of the invasion process. Extrapolation to zero force reveals single step times for DNA invading DNA four times faster than for RNA invading RNA. We also study the kinetics of DNA invading RNA, a process that in the absence of force would rarely occur. Our results reveal the importance of sequence effects for the TMSD process and have relevance for a wide range of applications in nucleic acid nanotechnology and synthetic biology.

Biomedical Utility of Non-Enzymatic DNA Amplification Reaction: From Material Design to Diagnosis and Treatment.

Nucleic acid nanotechnology has become a promising strategy for disease diagnosis and treatment, owing to remarkable programmability, precision, and biocompatibility. However, current biosensing and biotherapy approaches by nucleic acids exhibit limitations in sensitivity, specificity, versatility, and real-time monitoring. DNA amplification reactions present an advantageous strategy to enhance the performance of biosensing and biotherapy platforms. Non-enzymatic DNA amplification reaction (NEDAR), such as hybridization chain reaction and catalytic hairpin assembly, operate via strand displacement. NEDAR presents distinct advantages over traditional enzymatic DNA amplification reactions, including simplified procedures, milder reaction conditions, higher specificity, enhanced controllability, and excellent versatility. Consequently, research focusing on NEDAR-based biosensing and biotherapy has garnered significant attention. NEDAR demonstrates high efficacy in detecting multiple types of biomarkers, including nucleic acids, small molecules, and proteins, with high sensitivity and specificity, enabling the parallel detection of multiple targets. Besides, NEDAR can strengthen drug therapy, cellular behavior control, and cell encapsulation. Moreover, NEDAR holds promise for constructing assembled diagnosis-treatment nanoplatforms in the forms of pure DNA nanostructures and hybrid nanomaterials, which offer utility in disease monitoring and precise treatment. Thus, this paper aims to comprehensively elucidate the reaction mechanism of NEDAR and review the substantial advancements in NEDAR-based diagnosis and treatment over the past five years, encompassing NEDAR-based design strategies, applications, and prospects.

The unusual structural properties and potential biological relevance of switchback DNA

Synthetic DNA motifs form the basis of nucleic acid nanotechnology. The biochemical and biophysical properties of these motifs determine their applications. Here, we present a detailed characterization of switchback DNA, a globally left-handed structure composed of two parallel DNA strands. Compared to a conventional duplex, switchback DNA shows lower thermodynamic stability and requires higher magnesium concentration for assembly but exhibits enhanced biostability against some nucleases. Strand competition and strand displacement experiments show that component sequences have an absolute preference for duplex complements instead of their switchback partners. Further, we hypothesize a potential role for switchback DNA as an alternate structure in sequences containing short tandem repeats. Together with small molecule binding experiments and cell studies, our results open new avenues for switchback DNA in biology and nanotechnology.

Kinetic Proofreading Can Enhance Specificity in a Nonenzymatic DNA Strand Displacement Network.

Kinetic proofreading is used throughout natural systems to enhance the specificity of molecular recognition. At its most basic level, kinetic proofreading uses a supply of chemical fuel to drive a recognition interaction out of equilibrium, allowing a single free-energy difference between correct and incorrect targets to be exploited two or more times. Despite its importance in biology, there has been little effort to incorporate kinetic proofreading into synthetic systems in which molecular recognition is important, such as nucleic acid nanotechnology. In this article, we introduce a DNA strand displacement-based kinetic proofreading motif, showing that the consumption of a DNA-based fuel can be used to enhance molecular recognition during a templated dimerization reaction. We then show that kinetic proofreading can enhance the specificity with which a probe discriminates single nucleotide mutations, both in terms of the initial rate with which the probe reacts and the long-time behavior.

Tetrahedral DNA-Based Functional MicroRNA-21 Delivery System: Application to Corneal Epithelial Wound Healing.

Corneal injury occurs frequently which may lead to serious visual impairment. Rapid and efficient re-epithelialization after corneal epithelial injury is the key issue for maintaining corneal homeostasis. Among various treatment strategies, microRNA (miR)-based therapy shows great potential. However, structural limitations of miRNAs hinder its biomedical functionality. Nucleic acid nanotechnology is an appealing candidate for gene delivery because of its flexible modification and excellent biocompatibility. Herein, modified 3D tetrahedral framework nucleic acids (tFNAs) utilized as gene carriers for miR-21 delivery are constructed. TFNAs-miR-21 (T-21) shows great enzymatic resistance in extracellular environment and payload delivery into human corneal epithelial cells (HCECs) via clathrin-mediated endocytosis. T-21 facilitates proliferation and migration in HCECs via activating PI3K/AKT and ERK1/2 signaling pathways in vitro. In vivo studies, T-21 can be internalized by corneal epithelium in mice. In the mice corneal scratch model, T-21 ophthalmic solutions used as eye drops show no apparent side effects on the ocular surface histologically and exert great potential in accelerating corneal wound healing. These findings demonstrate that modified tFNAs are promising candidates for miRNA delivery for corneal wound healing. The convenient administration and great biocompatibility of tetrahedral DNA nanoparticles highlight its potential as gene transporter in solving ocular problems.

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.

Heterogeneous DNA hydrogel loaded with Apt02 modified tetrahedral framework nucleic acid accelerated critical-size bone defect repair

Segmental bone defects, stemming from trauma, infection, and tumors, pose formidable clinical challenges. Traditional bone repair materials, such as autologous and allogeneic bone grafts, grapple with limitations including source scarcity and immune rejection risks. The advent of nucleic acid nanotechnology, particularly the use of DNA hydrogels in tissue engineering, presents a promising solution, attributed to their biocompatibility, biodegradability, and programmability. However, these hydrogels, typically hindered by high gelation temperatures (∼46 °C) and high construction costs, limit cell encapsulation and broader application. Our research introduces a novel polymer-modified DNA hydrogel, developed using nucleic acid nanotechnology, which gels at a more biocompatible temperature of 37 °C and is cost-effective. This hydrogel then incorporates tetrahedral Framework Nucleic Acid (tFNA) to enhance osteogenic mineralization. Furthermore, considering the modifiability of tFNA, we modified its chains with Aptamer02 (Apt02), an aptamer known to foster angiogenesis. This dual approach significantly accelerates osteogenic differentiation in bone marrow stromal cells (BMSCs) and angiogenesis in human umbilical vein endothelial cells (HUVECs), with cell sequencing confirming their targeting efficacy, respectively. In vivo experiments in rats with critical-size cranial bone defects demonstrate their effectiveness in enhancing new bone formation. This innovation not only offers a viable solution for repairing segmental bone defects but also opens avenues for future advancements in bone organoids construction, marking a significant advancement in tissue engineering and regenerative medicine.

Aptamer-functionalized Nucleic Acid Nanotechnology for Biosensing, Bioimaging and Cancer Therapy

Nucleic acids have enabled to fabricate self-assemblies and perform dynamic operations. Among different functional nucleic acids, aptamers can specifically bind to wide range of targets including proteins, viral antigens, living...

RNase H-Driven crRNA Switch Circuits for Rapid and Sensitive Detection of Various Analytical Targets.

The clustered regularly interspaced short palindromic repeats (CRISPR/Cas12a) system has exhibited great promise in the rapid and sensitive molecular diagnostics for its trans-cleavage property. However, most CRISPR/Cas system-based detection methods are designed for nucleic acids and require target preamplification to improve sensitivity and detection limits. Here, we propose a generic crRNA switch circuit-regulated CRISPR/Cas sensor for the sensitive detection of various targets. The crRNA switch is engineered and designed in a blocked state but can be activated in the presence of triggers, which are target-induced association DNA to initiate the trans-cleavage activity of Cas12a for signal reporting. Additionally, RNase H is introduced to specifically hydrolyze RNA duplexed with the DNA trigger, resulting in the regeneration of the trigger to activate more crRNA switches. Such a combination provides a generic and sensitive strategy for the effective sensing of the p53 sequence, thrombin, and adenosine triphosphate. The design is incorporated with nucleic acid nanotechnology and extensively broadens the application scope of the CRISPR technology in biosensing.

DNA T-shaped crossover tiles for 2D tessellation and nanoring reconfiguration

DNA tiles serve as the fundamental building blocks for DNA self-assembled nanostructures such as DNA arrays, origami, and designer crystals. Introducing additional binding arms to DNA crossover tiles holds the promise of unlocking diverse nano-assemblies and potential applications. Here, we present one-, two-, and three-layer T-shaped crossover tiles, by integrating T junction with antiparallel crossover tiles. These tiles carry over the orthogonal binding directions from T junction and retain the rigidity from antiparallel crossover tiles, enabling the assembly of various 2D tessellations. To demonstrate the versatility of the design rules, we create 2-state reconfigurable nanorings from both single-stranded tiles and single-unit assemblies. Moreover, four sets of 4-state reconfiguration systems are constructed, showing effective transformations between ladders and/or rings with pore sizes spanning ~20 nm to ~168 nm. These DNA tiles enrich the design tools in nucleic acid nanotechnology, offering exciting opportunities for the creation of artificial dynamic DNA nanopores.

Unmodificated stepless regulation of CRISPR/Cas12a multi-performance.

As CRISPR technology is promoted to more fine-divided molecular biology applications, its inherent performance finds it increasingly difficult to cope with diverse needs in these different fields, and how to more accurately control the performance has become a key issue to develop CRISPR technology to a new stage. Herein, we propose a CRISPR/Cas12a regulation strategy based on the powerful programmability of nucleic acid nanotechnology. Unlike previous difficult and rigid regulation of core components Cas nuclease and crRNA, only a simple switch of different external RNA accessories is required to change the reaction kinetics or thermodynamics, thereby finely and almost steplessly regulating multi-performance of CRISPR/Cas12a including activity, speed, specificity, compatibility, programmability and sensitivity. In particular, the significantly improved specificity is expected to mark advance the accuracy of molecular detection and the safety of gene editing. In addition, this strategy was applied to regulate the delayed activation of Cas12a, overcoming the compatibility problem of the one-pot assay without any physical separation or external stimulation, and demonstrating great potential for fine-grained control of CRISPR. This simple but powerful CRISPR regulation strategy without any component modification has pioneering flexibility and versatility, and will unlock the potential for deeper applications of CRISPR technology in many finely divided fields.

Noncanonical Condensation of Nucleic Acid Chains by Hydrophobic Gold Nanocrystals.

Nucleic acid condensates are essential for various biological processes and have numerous applications in nucleic acid nanotechnology, gene therapy, and mRNA vaccines. However, unlike the in vivo condensation that is dependent on motor proteins, the in vitro condensation efficiency remains to be improved. Here, we proposed a hydrophobic interaction-driven mechanism for condensing long nucleic acid chains using atomically precise hydrophobic gold nanoclusters (Au NCs). We found that hydrophobic Au NCs could condense long single-stranded DNA or RNA to form composites of spherical nanostructures, which further assembled into bead-shaped suprastructures in the presence of excessive Au NCs. Thus, suprastructures displayed gel-like behaviors, and Au NCs could diffuse freely inside the condensates, which resemble the collective motions of condensin complexes inside chromosomes. The dynamic hydrophobic interactions between Au NCs and bases allow for the reversible release of nucleic acids in the presence of mild triggering agents. Our method represents a significant advancement toward the development of more efficient and versatile nucleic acid condensation techniques.

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.

Harnessing Nucleic Acids Nanotechnology for Bone/Cartilage Regeneration.

The effective regeneration of weight-bearing bone defects and critical-sized cartilage defects remains a significant clinical challenge. Traditional treatments such as autologous and allograft bone grafting have not been successful in achieving the desired outcomes, necessitating the need for innovative therapeutic approaches. Nucleic acids have attracted significant attention due to their ability to be designed to form discrete structures and programmed to perform specific functions at the nanoscale. The advantages of nucleic acid nanotechnology offer numerous opportunities for in-cell and in vivo applications, and hold great promise for advancing the field of biomaterials. In this review, the current abilities of nucleic acid nanotechnology to be applied in bone and cartilage regeneration are summarized and insights into the challenges and future directions for the development of this technology are provided.

Unravelling the Drug Encapsulation Ability of Functional DNA Origami Nanostructures: Current Understanding and Future Prospects on Targeted Drug Delivery.

Rapid breakthroughs in nucleic acid nanotechnology have always driven the creation of nano-assemblies with programmable design, potent functionality, good biocompatibility, and remarkable biosafety during the last few decades. Researchers are constantly looking for more powerful techniques that provide enhanced accuracy with greater resolution. The self-assembly of rationally designed nanostructures is now possible because of bottom-up structural nucleic acid (DNA and RNA) nanotechnology, notably DNA origami. Because DNA origami nanostructures can be organized precisely with nanoscale accuracy, they serve as a solid foundation for the exact arrangement of other functional materials for use in a number of applications in structural biology, biophysics, renewable energy, photonics, electronics, medicine, etc. DNA origami facilitates the creation of next-generation drug vectors to help in the solving of the rising demand on disease detection and therapy, as well as other biomedicine-related strategies in the real world. These DNA nanostructures, generated using Watson-Crick base pairing, exhibit a wide variety of properties, including great adaptability, precise programmability, and exceptionally low cytotoxicity in vitro and in vivo. This paper summarizes the synthesis of DNA origami and the drug encapsulation ability of functionalized DNA origami nanostructures. Finally, the remaining obstacles and prospects for DNA origami nanostructures in biomedical sciences are also highlighted.

Modular, Articulated Models of DNA and Peptide Nucleic Acids for Nanotechnology Education

ABSTRACT Dynamic and flexible nucleic acid models can provide current and future scientists with physical intuition for the structure of DNA and the ways that DNA and its synthetic mimics can be used to build self-assembling structures and advanced nanomachines. As more research labs and classrooms dive into the field of structural nucleic acid nanotechnology, students and researchers need access to interactive, dynamic, handheld models. Here, we present a 3D-printable kit for the construction of DNA and peptide nucleic acid (PNA). We have engineered a previous modular DNA kit to reduce costs while improving ease of assembly, flexibility, and robustness. We have also expanded the scope of available snap-together models by creating the first 3D-printable models of γPNA, an emerging material for nuclease- and protease-resistance nanotechnology. Building on previous research, representative nucleic acid duplexes were split into logical monomer segments, and atomic coordinates were used to create solid models for 3D printing. We used a human factors approach to customize 3 types of articulated snap-together connectors that allow for physically relevant motion characteristic of each interface in the model. Modules are easy to connect and separate manually but stay together when the model is manipulated. To greatly reduce cost, we bundled these segments for printing, and we created a miniaturized version that uses less than half the printing material to build. Our novel 3D-printed articulated snap-together models capture the flexibility and robustness of DNA and γPNA nanostructures. Resulting handheld helical models replicate the geometries in published structures and can now flex to form crossovers and allow biologically relevant zipping and unzipping to allow complex demonstrations of nanomachines undergoing strand displacement reactions. Finally, the same tools used to create these models can be readily applied to other types of backbones and nucleobases for endless research and education possibilities.

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.