DNA-based Nanostructures Research Articles

Programmable DNA-based nanostructure sensors for mutation detection coupled with asymmetric amplification in precision genome profiling

We introduce a DNA-based nanostructure sensor for the rapid, precise identification of single-nucleotide polymorphisms (SNPs), a key factor in determining disease susceptibility and tailoring medical treatment. The proposed two-state sensor design facilitates multiplexed, sequence-specific detection of double-stranded genomic DNA (gDNA), overcoming traditional obstacles presented by base pairing interference. Utilizing detector strands with binding penalties, the sensor achieves high SNP specificity, discernible by distinct gel electrophoresis band positions. Coupled with asymmetric PCR (asy-PCR), our integrated system amplifies genetic profiling effectiveness, especially in clinical biopsies, enhancing gDNA detection sensitivity and specificity for concurrent multiple mutation identifications. This cost-effective method ($0.41 per test) streamlines mutation screening, enabling high-throughput analysis with limited equipment—requiring only a single staining agent and a unified electrophoresis apparatus. Validated clinically with 70 colorectal cancer samples, the system boasts a 98.57% sensitivity and 95.71% specificity for KRAS mutations, surpassing traditional PCR detection and uncovering low-frequency mutations untraceable by direct sequencing. Our findings suggest this asy-PCR enhanced DNA-based nanostructure sensor system has the potential to revolutionize genetic diagnostics, offering expansive applications in detecting viral variants and microbial strains, thus advancing personalized genetic testing.

Directing Nanoparticle Organization in Response to Diverse Chemical Inputs.

Signaling cascades are crucial for transducing stimuli in biological systems, enabling multiple stimuli to regulate a downstream target with precisely controlled timing and amplifying signals through a series of intermediary reactions. Developing a robust signaling system with such capabilities would be pivotal for programming complex behaviors in synthetic DNA-based molecular devices. However, although "software" such as nucleic acid circuits could potentially be harnessed to relay signals to DNA-based nanostructure hardware, such explorations have been limited. Here, we develop a platform for transducing a variety of stimuli via messenger-mediated reactions to regulate the release and reloading of gold nanoparticles (AuNPs) in a 3D DNA framework. In the first step, an in vitro transcription circuit is engineered to sense and amplify chemical stimuli, including arbitrary DNA sequences and proteins, producing RNA. In the second step, the RNA releases the DNA-coated AuNPs from the DNA framework via a strand displacement reaction. AuNP reloading is controlled by a separate step driven by degradation of the RNA. Our platform holds promise for applications requiring dynamic multiagent control over DNA-based devices, offering a versatile tool for advanced molecular device engineering.

A bulged-type enzyme-free recognition strategy designed for single nucleotide polymorphisms integrating with label-free electrochemical biosensor

Compared to conventional nucleic acid detection methods, label-free single nucleotide polymorphism (SNP) detection presents challenging due to the necessity of discerning single base mismatches, especially in the field of enzyme-free detection. In this study, we introduce a novel bulged-type DNA duplex probe designed to significantly amplify single-base differences. This probe is integrated with programmable DNA-based nanostructures to develop a sensitive, label-free biosensor for nonenzymatic SNP detection. The duplex probe with one bulge could selectively identify wild-typed DNA (WT) and mutant-type DNA (MT) based on a competitive strand displacement reaction mechanism. The hyperbranched HCR (HHCR) by incorporating of hairpin DNA into the DNA tetrahedron and surface-tethering on the portable screen printing electrode (SPCE) significantly favor the formation of negatively charged DNA nanostructure. We harnessed strong repulsion of DNA nanostructure towards the electroactive [Fe(CN)₆]³⁻/⁴⁻ in combination with electrochemical technique to create a label-free biosensor. This simple, enzyme-free and label-free biosensor could detect MT with a detection limit of 56 aM, even in multiple sequence backgrounds. The study served as the proof-of-concept for the integration of enzyme-free competitive mechanism and label-free strategy, which can be extended as a powerful tool to various fields.

Binding Site Programmable Self-Assembly of 3D Hierarchical DNA Origami Nanostructures.

DNA nanotechnology has broad applications in biomedical drug delivery and programmable materials. Characterization of the self-assembly of DNA origami and quantum dots (QDs) is necessary for the development of new DNA-based nanostructures. We use computation and experiment to show that the self-assembly of 3D hierarchical nanostructures can be controlled by programming the binding site number and their positions on DNA origami. Using biotinylated pentagonal pyramid wireframe DNA origamis and streptavidin capped QDs, we demonstrate that DNA origami with 1 binding site at the outer vertex can assemble multimeric origamis with up to 6 DNA origamis on 1 QD, and DNA origami with 1 binding site at the inner center can only assemble monomeric and dimeric origamis. Meanwhile, the yield percentages of different multimeric origamis are controlled by the QD:DNA-origami stoichiometric mixing ratio. DNA origamis with 2 binding sites at the αγ positions (of the pentagon) make larger nanostructures than those with binding sites at the αβ positions. In general, increasing the number of binding sites leads to increases in the nanostructure size. At high DNA origami concentration, the QD number in each cluster becomes the limiting factor for the growth of nanostructures. We find that reducing the QD size can also affect the self-assembly because of the reduced access to the binding sites from more densely packed origamis.

Synthetic DNA-based Swimmers Driven by Enzyme Catalysis.

Here, we report DNA-based synthetic nanostructures decorated with enzymes (hereafter referred to as DNA-enzyme swimmers) that self-propel by converting the enzymatic substrate to the product in solution. The DNA-enzyme swimmers are obtained from tubular DNA structures that self-assemble spontaneously by the hybridization of DNA tiles. We functionalize these DNA structures with two different enzymes, urease and catalase, and show that they exhibit concentration-dependent movement and enhanced diffusion upon addition of the enzymatic substrate (i.e., urea and H2O2). To demonstrate the programmability of such DNA-based swimmers, we also engineer DNA strands that displace the enzyme from the DNA scaffold, thus acting as molecular "brakes" on the DNA swimmers. These results serve as a first proof of principle for the development of synthetic DNA-based enzyme-powered swimmers that can self-propel in fluids.

DNA-based nanostructures for RNA delivery.

RNA-based therapeutics have emerged as a promising approach for the treatment of various diseases, including cancer, genetic disorders, and infectious diseases. However, the delivery of RNA molecules into target cells has been a major challenge due to their susceptibility to degradation and inefficient cellular uptake. To overcome these hurdles, DNA-based nano technology offers an unprecedented opportunity as a potential delivery platform for RNA therapeutics. Due to its excellent characteristics such as programmability and biocompatibility, these DNA-based nanostructures, composed of DNA molecules assembled into precise and programmable structures, have garnered significant attention as ideal building materials for protecting and delivering RNA payloads to the desired cellular destinations. In this review, we highlight the current progress in the design and application of three DNA-based nanostructures: DNA origami, lipid-nanoparticle (LNP) technology related to frame guided assembly (FGA), and DNA hydrogel for the delivery of RNA molecules. Their biomedical applications are briefly discussed and the challenges and future perspectives in this field are also highlighted.

Double-Stranded DNA Immobilized in Lying-Flat and Upright Orientation on a PNIPAm-Coated Surface: A Theoretical Study.

Surface-immobilized double-stranded DNA (dsDNA) in upright orientation plays an important role in optimizing and understanding DNA-based nanosensors and nanodevices. However, it is difficult to regulate the surface density of upright DNA due to the fact that DNA usually stands vertically at a high packing density but may lie down at a low packing density. We herein report dsDNA immobilized in upright orientation on a poly(N-isopropylacrylamide) (PNIPAm)-coated surface in theory. The theoretical results reveal that the angle of upright DNA relative to the surface is larger than that of DNA immobilized on the bare surface caused by the lying-flat DNA under proper PNIPAm surface coverage at 45 °C. The surface density of upright DNA is significantly influenced by DNA concentration and DNA length. It is envisioned that the density-regulated DNA molecules immobilized in upright orientation in the present work are well suited to bottom-up construction of complex DNA-based nanostructures and nanodevices.

Assessing the influence of small structural modifications in simple DNA-based nanostructures on their role as drug nanocarriers.

DNA nanotechnology leverages Watson-Crick-Franklin base-pairing interactions to build complex DNA-based nanostructures (DNS). Due to DNA specific self-assembly properties, DNS can be designed with a total control of their architecture, which has been demonstrated to have an impact on the overall DNS features. Indeed, structural properties such as the shape, size and flexibility of DNS can influence their biostability as well as their ability to internalise into cells. We present here two series of simple DNS with small and precise variations related to their length or flexibility and study the influence that these structural changes have on their overall properties as drug nanocarriers. Results indicate that shorter and more flexible DNS present higher stability towards nuclease degradation. These structural changes also have a certain effect on their cell internalisation ability and drug release rate. Consequently, drug-loaded DNS cytotoxicity varies according to the design, with lower cell viability values obtained in the DNS exhibiting faster drug release and larger cell interaction rates. In summary, small changes in the structure of simple DNS can have an influence on their overall capabilities as drug nanocarriers. The effects reported here could guide the design of simple DNS for future therapeutic uses.

DNA-modulated dimerization and oligomerization of cell membrane receptors.

DNA-based nanostructures and nanodevices have recently been employed for a broad range of applications in modulating the assemblies and interaction patterns of different cell membrane receptors. These versatile nanodevices can be rationally designed with modular structures, easily programmed and tweaked such that they may act as smart chemical biology and cell biology tools to reveal insights into complicated cellular signaling processes. Their outstanding in vitro and cellular features have also begun to be further validated for some in vivo applications and demonstrated their great biomedical potential. In this review, we will highlight some key current advances in the molecular engineering and biological applications of DNA-based functional nanodevices, with a focus on how these tools have been used to respond and modulate membrane receptor dimerizations and/or oligomerizations, as a way to control cellular signaling processes. Some current challenges and future directions to further develop and apply these DNA nanodevices will also be discussed.

Structural DNA nanotechnology at the nexus of next-generation bio-applications: challenges and perspectives.

DNA nanotechnology has significantly progressed in the last four decades, creating nucleic acid structures widely used in various biological applications. The structural flexibility, programmability, and multiform customization of DNA-based nanostructures make them ideal for creating structures of all sizes and shapes and multivalent drug delivery systems. Since then, DNA nanotechnology has advanced significantly, and numerous DNA nanostructures have been used in biology and other scientific disciplines. Despite the progress made in DNA nanotechnology, challenges still need to be addressed before DNA nanostructures can be widely used in biological interfaces. We can open the door for upcoming uses of DNA nanoparticles by tackling these issues and looking into new avenues. The historical development of various DNA nanomaterials has been thoroughly examined in this review, along with the underlying theoretical underpinnings, a summary of their applications in various fields, and an examination of the current roadblocks and potential future directions.

Self-assembled methodologies for the construction of DNA nanostructures and biological applications.

Over the past decades, deoxyribonucleic acid (DNA), as a versatile building block, has been widely employed to construct functionalized nanostructures. Among the diverse types of materials, DNA related nanostructures have gained growing attention due to their intrinsic programmability, favorable biocompatibility, and strong molecular recognition capability. The conventional construction strategy for building DNA structures is based on Watson-Crick base-pairing rules, which are mainly driven by the hydrogen bonding of bases. However, hydrogen bonding-based DNA nanostructures cannot meet the requirements of specific morphology and multifunctionality. Currently, various functional elements have been introduced to expand the synthetic methodologies for constructing the DNA hybrid nanostructures, including small molecules, peptide polymers, organic ligands and transition metal ions. Besides, the potential applications for these DNA hybrid nanostructures have also been explored. It has been demonstrated that DNA hybrid structures with various properties can be extensively applied in the fields of magnetic resonance, luminescence imaging, biomedical detection, and drug delivery systems. In this review, we highlight the pioneering contributions to the methodologies of DNA-based nanostructure assembly. Furthermore, the recent advances in drug delivery systems and biomedical diagnosis based on DNA hybrid nanostructures are briefly summarized.

PH-responsive i-motif-conjugated nanoparticles for MRI analysis.

Gadolinium (Gd)-based contrast agents (CAs) are widely used to enhance anatomical details in magnetic resonance imaging (MRI). Significant research has expanded the field of CAs into bioresponsive CAs by modulating the signal to image and monitor biochemical processes, such as pH. In this work, we introduce the modular, dynamic actuation mechanism of DNA-based nanostructures as a new way to modulate the MRI signal based on the rotational correlation time, τR. We combined a pH-responsive oligonucleotide (i-motif) and a clinical standard CA (Gd-DOTA) to develop a pH-responsive MRI CA. The i-motif folds into a quadruplex under acidic conditions and was incorporated onto gold nanoparticles (iM-GNP) to achieve increased relaxivity, r1, compared to the unbound i-motif. In vitro, iM-GNP resulted in a significant increase in r1 over a decreasing pH range (7.5-4.5) with a calculated pKa = 5.88 ± 0.01 and a 16.7% change per 0.1 pH unit. In comparison, a control CA with a non-responsive DNA strand (T33-GNP) did not show a significant change in r1 over the same pH range. The iM-GNP was further evaluated in 20% human serum and demonstrated a 28.14 ± 11.2% increase in signal from neutral pH to acidic pH. This approach paves a path for novel programmable, dynamic DNA-based complexes for τR-modulated bioresponsive MRI CAs.

3DNA: A Tool for Sculpting Brick-Based DNA Nanostructures

To assist in the speed and accuracy of designing brick-based DNA nanostructures, we introduce a lightweight software suite 3DNA that can be used to generate complex structures. Currently, implementation of this fabrication strategy involves working with generalized, typically commercial CAD software, ad-hoc sequence-generating scripts, and visualization software, which must often be integrated together with an experimental lab setup for handling the hundreds or thousands of constituent DNA sequences. 3DNA encapsulates the solutions to these challenges in one package by providing a customized, easy-to-use molecular canvas and back-end functionality to assist in both visualization and sequence design. The primary motivation behind this software is enabling broader use of the brick-based method for constructing rigid, 3D DNA-based nanostructures, first introduced in 2012. 3DNA is developed to provide a streamlined, real-time workflow for designing and implementing this type of 3D nanostructure by integrating different visualization and design modules. Due to its cross-platform nature, it can be used on the most popular desktop environments, i.e., Windows, Mac OS X, and various flavors of Linux. 3DNA utilizes toolbar-based navigation to create a user-friendly GUI and includes a customized feature to analyze the constituent DNA sequences. Finally, the oligonucleotide sequences themselves can either be created on the fly by a random sequence generator, or selected from a pre-existing set of sequences making up a larger molecular canvas.

Defined covalent attachment of three cancer drugs to DNA origami increases cytotoxicity at nanomolar concentration

DNA nanostructures have captured great interest as drug delivery vehicles for cancer therapy. Despite rapid progress in the field, some hurdles, such as low cellular uptake, low tissue specificity or ambiguous drug loading, remain unsolved. Herein, well-known antitumor drugs (doxorubicin, auristatin, and floxuridine) were site-specifically incorporated into DNA nanostructures, demonstrating the potential advantages of covalently linking drug molecules via structural staples instead of incorporating the drugs by noncovalent binding interactions. The covalent strategy avoids critical issues such as an unknown number of drug-DNA binding events and premature drug release. Moreover, covalently modified origami offers the possibility of precisely incorporating several synergetic antitumor drugs into the DNA nanostructure at a predefined molar ratio and to control the exact spatial orientation of drugs into DNA origami. Additionally, DNA-based nanoscaffolds have been reported to have a low intracellular uptake. Thus, two cellular uptake enhancing mechanisms were studied: the introduction of folate units covalently linked to DNA origami and the transfection of DNA origami with Lipofectamine. Importantly, both methods increased the internalization of DNA origami into HTB38 and HCC2998 colorectal cancer cells and produced greater cytotoxic activity when the DNA origami incorporated antiproliferative drugs. The results here present a successful and conceptually distinct approach for the development of DNA-based nanostructures as drug delivery vehicles, which can be considered an important step towards the development of highly precise nanomedicines.

Leveraging DNA-Based Nanostructures for Advanced Error Detection and Correction in Data Communication.

This study demonstrates the implementation of the Hamming code using DNA-based nanostructures for error detection and correction in communication systems. The designed DNA nanostructures conduct logical operations to compute check codes and identify and correct erroneous data based on fluorescence signals. The execution of intricate DNA logic operations requires individuals with specialized training. By interpretation of the fluorescence signals generated by the DNA nanostructures, binary language can be extracted, effectively protecting data security. The findings highlight the potential of DNA as a versatile platform for reliable data transmission.

2′-Fluoro-substituted DNA-induced self-assembly strategy for engineering hybrid nanospheres

The physicochemical differences between DNA and other molecules pose a challenge to the construction of DNA-based nanostructures. Herein, we propose a straightforward approach for preparing multifunctional DNA-based nanospheres through direct self-assembly of 2′-fluoro-substituted single-stranded DNA (2′F-DNA) with various small molecules. Molecular dynamics simulation revealed that 2′F substitution in DNA can cause the repulsion of adjacent PO43- group, leading to local stretching of the DNA structure. Moreover, 2′F substituent induced the regular polarization of H2O nearby F to form the hydration layer, which interrupts inherent interactions among bases. In this way, the bases of 2′F-DNA chain have fewer constraints and more flexibility in conformation, facilitating their non-covalent interactions with other molecules and enhancing the self-assembly capacity of 2′F-DNA. Consequently, 2′F-DNA can bind to more molecules, tending to spontaneously form hybrid DNA nanospheres. Following this approach, a chemo-gene therapy 2′F-DNA/doxorubicin model was designed, showing the significant synergistic anti-tumor therapeutic efficacy. Taken together, this study provides an expandable approach for constructing engineering hybrid nanospheres using DNA and other small molecules, which holds great potential for further biological applications.

Caffeine-induced release of small molecules from DNA nanostructures

Several planar aromatic molecules are known to intercalate between base pairs of double-stranded DNA. This mode of interaction has been used to stain DNA as well as to load drug molecules onto DNA-based nanostructures. Some small molecules are also known to induce deintercalation in double-stranded DNA, one such molecule being caffeine. Here, we compared the ability of caffeine to cause deintercalation of ethidium bromide, a representative DNA intercalator, from duplex DNA and three DNA motifs of increasing structural complexity (four-way junction, double crossover motif, and DNA tensegrity triangle). We found that caffeine impedes the binding of ethidium bromide in all these structures to the same extent, with some differences in deintercalation profiles. Our results can be useful in the design of DNA nanocarriers for intercalating drugs, where drug release can be chemically stimulated by other small molecules.

A Dynamic Control Center Based on a DNA Reaction Network for Programmable Building of DNA Nanostructures.

DNA-based nanostructures allow for complex self-assembly with nanometer precision through the specificity of Watson-Crick base pairing, but network behavior-directed control of the kinetic process is less studied. Here we show how the DNA reaction network (DRN), which has emerged as a reliable and programmable way to implement artificial network dynamics, can be built as the control center of programmable nanostructures, allowing spatiotemporal control over the dynamic behavior of DNA nanotubes. We chose a common network motif in biological control systems, the feed-forward loop, as the model network and demonstrated that dynamic behaviors, such as self-tuning control and multilayer hierarchical assembly, could be programmed by constructing an inhibition network and an excitation network, separately, in buffer solution and inside protocells.

DNA‐Encoded Synthetic Systems: Coding More than Life

DNA‐Encoded Synthetic Systems: Coding More than Life

A DNA-based nanodevice for near-infrared light-controlled drug release and bioimaging

DNA-based nanostructures have shown curative potential in drug delivery and anti-tumor therapy. However, the applications of such systems are limited by the lack of precise control over the space and duration of drug release. Here, we report a near-infrared (NIR) light-guided DNA nanodevice that enables controlled drug release for precise tumor imaging and therapy. The DNA nanodevice is constructed by engineering GC-rich DNA duplexes with an ultraviolet (UV) light labile moiety and further modification onto the surface of upconversion nanoparticles (UCNPs). The chemotherapeutic drug (doxorubicin, Dox) is intercalated into the GC motif of DNA duplexes, where the fluorescence signal of Dox is markedly quenched. Upon NIR light irradiation, the UCNPs acting as energy transducers emit UV light that can break the photolabile moiety in DNA duplexes, resulting in the controlled release of Dox and recovery of their fluorescence signal. We present that this DNA nanodevice is not only able to selectively induce tumor cells apoptosis via NIR light-mediated activation, but also enables in situ imaging and monitoring of drug release. Therefore, this modular strategy opens a window for remotely controlled drug delivery.