Magnetic separation coupled with gold nanoparticle-assisted hybridization chain reaction for simultaneous detection of exomiR-21 and exomiR-155.

Fullerene-based PEC aptasensor with DNA super sandwich structures and Pb2+-G quadruplex structures

DNA nanotechnology enables the precise construction of intricate nanoscale structures. Over the past two decades, significant progress has been made in incorporating dynamic functionalities into these nanostructures. Concurrently, innovative strategies have emerged for their self-assembly and surface patterning into larger, more complex architectures. This review explores the convergence of these two key capabilities-reconfigurability and hierarchical assembly-to engineer DNA origami superstructures with intrinsic dynamic behavior. We begin by outlining foundational strategies in dynamic design, hierarchical assembly, and surface placement, then review recent progress in leveraging these strategies to construct dynamic superstructures with emergent behaviors. The article concludes with a roadmap of major challenges and opportunities shaping the future of this rapidly evolving field.

Light-dependent on/off ratio of organic photoelectrochemical transistor biosensing

DNA-templated metallization has emerged as an efficient strategy for creating nanoscale-metal DNA hybrid structures with a desirable conformation and function. Despite the potential of DNA-metal hybrids, their use as combinatory therapeutic agents has rarely been examined. Herein, we present a simple approach for fabricating a multipurpose DNA superstructure that serves as an efficient photoimmunotherapy agent. Specifically, we adsorb and locally concentrate Au ions onto DNA superstructures through induced local reduction, resulting in the formation of Au nanoclusters. The mechanical and optical properties of these metallic nanoclusters can be rationally controlled by their conformations and metal ions. The resulting golden DNA superstructures (GDSs) exhibit significant photothermal effects that induce cancer cell apoptosis. When sequence-specific immunostimulatory effects of DNA are combined, GDSs provide a synergistic effect to eradicate cancer and inhibit metastasis, demonstrating potential as a combinatory therapeutic agent for tumor treatment. Altogether, the DNA superstructure-templated metal casting system offers promising materials for future biomedical applications.

Abstract Inspired by intrinsically disordered proteins in nature, DNA aptamers can be engineered to display strongly homotropic allosteric (or cooperative) ligand binding, representing a unique feature that could be of great utility in applications such as biosensing, imaging and drug delivery. The use of an intrinsic disorder mechanism, however, comes with an inherent drawback of significantly reduced overall binding affinity. We hypothesize that it could be addressed via the design of multivalent supramolecular aptamers. We built functional DNA superstructures (denoted as 3D DNA), made of long‐chain DNA containing tandem repeating DNA aptamers (or concatemeric aptamers). The 3D DNA systems exhibit highly cooperative binding to both small molecules and proteins, without loss of binding affinities of their parent aptamers. We further produced a highly responsive sensor for fluorescence imaging of glutamate stimulation‐evoked adenosine triphosphate (ATP) release in neurons, as well as force stimulus‐triggered ATP release in astrocytes.

Inspired by intrinsically disordered proteins in nature, DNA aptamers can be engineered to display strongly homotropic allosteric (or cooperative) ligand binding, represents a unique feature that could be of great utility in applications such as biosensing, imaging and drug delivery. The use of intrinsic disorder mechanism, however, comes with an inherent drawback: the overall binding affinity is significantly reduced. We hypothesize that this issue could be addressed via the design of multivalent supramolecular aptamers. To confirm this idea, we engineered a DNA aptameric assembly, which we denote "functional DNA superstructures" (3D DNA). This functional 3D DNA is made of long chain DNA containing tandem repeating DNA aptamers (or concatemeric aptamers). We demonstrate that functional 3D DNA systems exhibit highly cooperative binding to both small molecules and proteins, without loss of binding affinities of their parent aptamers. We further use this to produce a highly responsive sensor for fluorescence imaging of glutamate stimulation-evoked adenosine triphosphate (ATP) release in neurons, as well as force stimulus-triggered ATP release in astrocytes.

Real-time monitoring of different types of intracellular tumor-related biomarkers is of key importance for the identification of tumor cells. However, it is hampered by the low abundance of biomarkers, inefficient free diffusion of reactants, and complex cytoplasmic milieu. Herein, we present a stable and general method for in situ imaging of microRNA-21 and telomerase utilizing simple highly integrated dual tetrahedral DNA nanostructures (TDNs) that can naturally enter cells, which could initiate to form the three-dimensional (3D) higher-order DNA superstructures (DNA nanofireworks, DNFs) through a reliable target-triggered entropy-driven strand displacement reaction in living cells for remarkable signal amplification. Importantly, the excellent biostability, biocompatibility, and sensitivity of this approach benefited from (i) the precise multidirectional arrangement of probes with a pure DNA structure and (ii) the local target concentration enhanced by the spatially confined microdomain inside the DNFs. This strategy provides a pivotal molecular toolbox for broad applications such as biomedical imaging and early precise cancer diagnosis.

We designed a paper-based analytical device by integrating horseradish peroxidase (HRP)-encapsulated 3D DNA for visual detection of alkaline phosphatase (ALP). This device allows on-paper sample pre-treatment, target recognition and signal readout, enabling simple (without additional pre-treatment of blood samples) and rapid (within 23 min) determination of ALP in clinical samples.

Nature makes use of molecular charges to operate specific biological synthesis and reactions. Targeting advanced opto-bioelectronic sensors, organic photoelectrochemical transistors (OPECTs), taking advantage of the light fuel substituting an external gate potential, is now debuting and expected to serve as a universal platform for studying the rich light-biomatter interplay for new bioanalytics. Given the ubiquity of charged biomolecules in nature, molecular charge manipulation should underpin a generic route for innovative OPECT regulation and operation, which nevertheless has remained unachieved. Herein, this work manifests the biological tuning of surface charge toward the OPECT biosensor, which was exemplified by a light-sensitive CdS quantum dot (QD) gate electrode interfaced by a smart DNA superstructure with adenosine triphosphate (ATP) responsiveness. Highly negative-charged supramolecular DNA concatemers were self-assembled via sequential hybridization, and the ATP-triggered disassembly of the DNA concatemers would cause a tandem change of the effective gate voltage and transfer characteristics with significantly improved resolution. The present opto-bioelectronic device translates the events of charged molecules into amplified electrical signals and outlines a generic format for the future exploitation of rich biological tunability and light-biomatter interplay for innovative bioanalytics and beyond.

CRISPR-Cas12a-activated palindrome-catalytic hairpin assembly for ultrasensitive fluorescence detection of HIV-1 DNA

DNA origami technology enables the folding of DNA strands into complex nanoscale shapes whose properties and interactions with molecular species often deviate significantly from that of genomic DNA. Here, we investigate the salting-out of different DNA origami shapes by the kosmotropic salt ammonium sulfate that is routinely employed in protein precipitation. We find that centrifugation in the presence of 3 M ammonium sulfate results in notable precipitation of DNA origami nanostructures but not of double-stranded genomic DNA. The precipitated DNA origami nanostructures can be resuspended in ammonium sulfate-free buffer without apparent formation of aggregates or loss of structural integrity. Even though quasi-1D six-helix bundle DNA origami are slightly less susceptible toward salting-out than more compact DNA origami triangles and 24-helix bundles, precipitation and recovery yields appear to be mostly independent of DNA origami shape and superstructure. Exploiting the specificity of ammonium sulfate salting-out for DNA origami nanostructures, we further apply this method to separate DNA origami triangles from genomic DNA fragments in a complex mixture. Our results thus demonstrate the possibility of concentrating and purifying DNA origami nanostructures by ammonium sulfate-induced salting-out.

Massive DNA testingrequires novel technologies to support a sustainablehealth system. In recent years, DNA superstructures have emerged asalternative probes and transducers. We, herein, report a multiplexedand highly sensitive approach based on an allele-specific hybridizationchain reaction (AS-HCR) in the array format to detect single-nucleotidevariants. Fast isothermal amplification was developed before activatingthe HCR process on a chip to work with genomic DNA. The assay principlewas demonstrated, and the variables for integrating the AS-HCR processand smartphone-based detection were also studied. The results werecompared to a conventional polymerase reaction chain (PCR)-based test.The developed multiplex method enabled higher selectivity againstsingle-base mismatch sequences at concentrations as low as 103 copies with a limit of detection of 0.7% of the mutant DNApercentage and good reproducibility (relative error: 5% for intra-assayand 17% for interassay). As proof of concept, the AS-HCR method wasapplied to clinical samples, including human cell cultures and biopsiedtissues of cancer patients. Accurate identification of single-nucleotidemutations in KRAS and NRAS geneswas validated, considering those obtained from the reference sequencingmethod. To conclude, AS-HCR is a rapid, simple, accurate, and cost-effectiveisothermal method that detects clinically relevant genetic variantsand has a high potential for point-of-care demands.

Substituent effect of benzyl moiety in nitroquinoxaline small molecules upon DNA binding: Cumulative destacking of DNA nucleobases leading to histone eviction

The formation of guanine quadruplexes (GQ) in DNA is crucial in telomere homeostasis and regulation of gene expression. Pollution metals can interfere with these DNA superstructures upon coordination. In this work, we study the affinity of the internal GQ channel site towards alkaline earth metal (Mg2+, Ca2+, Sr2+, and Ba2+), and (post‐)transition metal (Zn2+, Cd2+, Hg2+, and Pb2+) cations using density functional theory computations. We find that divalent cations generally bind to the GQ cavity with a higher affinity than conventional monovalent cations (e. g. K+). Importantly, we establish the nature of the cation‐GQ interaction and highlight the relationship between ionic and nuclear charge, and the electrostatic and covalent interactions. The covalent interaction strength plays an important role in the cation affinity and can be traced back to the relative stabilization of cations’ unoccupied atomic orbitals. Overall, our findings contribute to a deeper understanding of how pollution metals could induce genomic instability.

The structure and function of bacterial chromosomes are dynamically regulated by a wide variety of nucleoid-associated proteins (NAPs) and DNA superstructures, such as DNA supercoiling. In Escherichia coli, integration host factor (IHF), a NAP, binds to specific transcription promoters and regulatory DNA elements of DNA replication such as the replication origin oriC: binding to these elements depends on the cell cycle but underlying mechanisms are unknown. In this study, we combined GeF-seq (genome footprinting with high-throughput sequencing) with synchronization of the E. coli cell cycle to determine the genome-wide, cell cycle-dependent binding of IHF with base-pair resolution. The GeF-seq results in this study were qualified enough to analyze genomic IHF binding sites (e.g., oriC and the transcriptional promoters of ilvG and osmY) except some of the known sites. Unexpectedly, we found that before replication initiation, oriC was a predominant site for stable IHF binding, whereas all other loci exhibited reduced IHF binding. To reveal the specific mechanism of stable oriC–IHF binding, we inserted a truncated oriC sequence in the terC (replication terminus) locus of the genome. Before replication initiation, stable IHF binding was detected even at this additional oriC site, dependent on the specific DnaA-binding sequence DnaA box R1 within the site. DnaA oligomers formed on oriC might protect the oriC–IHF complex from IHF dissociation. After replication initiation, IHF rapidly dissociated from oriC, and IHF binding to other sites was sustained or stimulated. In addition, we identified a novel locus associated with cell cycle-dependent IHF binding. These findings provide mechanistic insight into IHF binding and dissociation in the genome.

There is currently a great need for developing a simple and effective biosensing platform for the detection of single biomolecules (e.g., DNAs, RNAs, or proteins) in the biological, medical, and environmental fields. Here, we show a versatile and sensitive fluorescence counting strategy for quantifying proteins and microRNAs by employing functional DNA superstructures (denoted as 3D DNA). A 3D DNA biolabel was first engineered to become highly fluorescent and carry recognition elements for the target of interest. The presence of a target cross-links the resultant of the 3D DNA biolabel and a surface-bound capturing antibody or DNA oligonucleotide, thus forming a sandwich complex that can be easily resolved using traditional fluorescence microscopy. The broad utility of this platform is illustrated by engineering two different 3D DNA biolabels that enable the quantification of β-lactamase (one secreted bacterial hydrolase) and miR-21 (one overexpressed microRNA in cancer cells) with detection limits of 100 aM and 1 fM, respectively. We envision that the approach described herein will find useful applications in chemical biology, medical diagnostics, and biosensing.

A 153-mer target DNA was amplified using ethynyl ferrocene dATP and a tailed forward primer resulting in a duplex with a single-stranded DNA tail for hybridization to a surface-tethered probe. A thiolated probe containing the sequence complementary to the tail as well as a 15 polythimine vertical spacer with a (CH2)6 spacer was immobilized on the surface of a gold electrode and hybridized to the ferrocene-modified complementary strand. Potential step chronoamperometry and cyclic voltammetry were used to probe the potential of zero charge, PZC, and the rate of heterogeneous electron transfer between the electrode and the immobilized ferrocene moieties. Chronoamperometry gives three, well-resolved exponential current–time decays corresponding to ferrocene centers located within 13 Å (4 bases) along the duplex. Significantly, the apparent standard heterogeneous electron transfer rate constant, kappo, observed depends on the initial potential, i.e., the rate of electron transfer at zero driving force is not the same for oxidation and reduction of the ferrocene labels. Moreover, the presence of ions, such as Sr2+, that strongly ion pair with the negatively charged DNA backbone modulates the electron transfer rate significantly. Specifically, kappo = 246 ± 23.5 and 14 ± 1.2 s–1 for reduction and oxidation, respectively, where the Sr2+ concentration is 10 mM, but the corresponding values in 1 M Sr2+ are 8 ± 0.8 and 150 ± 12 s–1. While other factors may be involved, these results are consistent with a model in which a low Sr2+ concentration and an initial potential that is negative of the PZC lead to electrostatic repulsion of the negatively charged DNA backbone and the negatively charged electrode. This leads to the DNA adopting an extended configuration (concertina open), resulting in a slow rate of heterogeneous electron transfer. In contrast, for ferrocene reduction, the initial potential is positive of PZC and the negatively charged DNA is electrostatically attracted to the electrode (concertina closed), giving a shorter electron transfer distance and a higher rate of heterogeneous electron transfer. When the Sr2+ concentration is high, the charge on the DNA backbone is compensated by the electrolyte and the charge on the electrode dominates the electron transfer dynamics and the opposite potential dependence is observed. These results open up the possibility of electromechanical switching using DNA superstructures.

Strategies that speed up the on-command release of proteins (e.g., enzymes) from stimuli-responsive materials are intrinsically necessary for biosensing applications, such as point-of-care testing, as they will achieve fast readouts with catalytic signal-amplification. However, current systems are challenging to work with because they usually exhibit response times on the order of hours up to days. Herein, we report on the first effort to construct a fast-responding gating system using protein-encapsulating functional DNA superstructures (denoted as protein@3D DNA). Proteins were directly embedded into 3D DNA during the one-pot rolling circle amplification process. We found that the specific DNA–DNA interaction and aptamer–ligand interaction could act as general protocols to release the loaded proteins from 3D DNA. The resulting gating system exhibits fast release kinetics on the order of minutes. Taking advantage of this finding, we designed a simple paper device by employing protein@3D DNA for colorimetric detection of toxin B (Clostridium difficile marker). This device is capable of detecting 0.1 nM toxin B within 16 minutes.

High-copy DNA repeats are building blocks of complex DNA superstructures. It has not been a trivial task to make a short DNA sequence elongated by simply repeating itself. In article number 2003671, Xin Wang and Xuerui Yang present a simple method named BPRE, which now makes mass production of the high-copy DNA repeats fast, cheap, and clean. As a typical application, reannealing of the DNA repeats generates elastic hydrogels with high capacity of drug absorption and prolonged release.