DNA Nanomachines Research Articles

Endogenous enzyme-activated AND-gate DNA nanomachines for intracellular miRNA detection and cell-selective imaging

The occurrence and development of tumors are accompanied by the abnormal expression of specific microRNAs (miRNAs). Therefore, miRNAs are considered as an important biomarker. The establishment of efficient, simple and sensitive miRNA imaging methods in living cells will contribute to the early diagnosis, treatment and drug development of diseases. In this study, we developed an endogenous enzyme-initiated AND logic circuit using gold nanocubes (AuNCs) as carriers for simultaneous detection of miRNA-21 and miRNA-210 in cells. Apurinic/apyrimidinic endonuclease 1 (APE1) and telomerase (TE), which are overexpressed in cancer cells, act as control switches in a logic circuit that enables sensitive in situ analysis of intracellular miRNAs without additional external intervention. At the same time, due to the lack of necessary enzymes as activation switches, the DNA circuit in normal cells remains in an inactive state. This strategy effectively reduces the risk of false positive signal generation. Our research results show that the logic circuit can not only distinguish between cancer cells and normal cells, and able to distinguish between different types of cancer cells. This finding provides a promising approach to accurately identify cell types.

Effective Enrichment of Free Radicals through Nanoconfinement Boosts Electrochemiluminescence of Carbon Dots Derived from Luminol.

Local enrichment of free radicals at the electrode interface may open new opportunities for the development of electrochemiluminescence (ECL) applications. The sensing platform was constructed by assembling ECL-emitting luminol derived carbon dots (Lu CDs) onto the heterojunction Tungsten disulfide/Covalent organic frameworks (WS2@COF) for the first time, establishing a nanoconfinement-reactor with significantly heightened ECL intensity and stability compared to the Lu CDs-H2O2 system. This enhanced performance is credited to the COF domain's restricted pore environment, where WS2@COF exhibits a more negative adsorption energy for H2O2, effectively enriching H2O2 in the catalytic edge sites of WS2. Furthermore, the internal electric field at the WS2 and COF interface accelerates electron flow, boosting WS2's catalytic activity and achieving domain-limited catalytic enhancement of ECL. Self-designed DNA nanomachines combined with cascading molecular keypad locking mechanisms are integrated into the biosensors, effectively guaranteeing the accuracy of the sensing process while providing crucial safeguards for molecular diagnostics and information security applications. In essence, this innovative approach represents the first system to enhance local free radical concentrations by enriching co-reactants on the electrode surface through nanoconfinement catalysis, yielding heightened ECL luminescence intensity. The potential impact of this novel strategy and sensing mechanism on real-bioanalysis applications is promising.

Effective Enrichment of Free Radicals through Nanoconfinement Boosts Electrochemiluminescence of Carbon Dots Derived from Luminol

Local enrichment of free radicals at the electrode interface may open new opportunities for the development of electrochemiluminescence (ECL) applications. The sensing platform was constructed by assembling ECL‐emitting luminol derived carbon dots (Lu CDs) onto the heterojunction Tungsten disulfide/Covalent organic frameworks (WS2@COF) for the first time, establishing a nanoconfinement‐reactor with significantly heightened ECL intensity and stability compared to the Lu CDs‐H2O2 system. This enhanced performance is credited to the COF domain's restricted pore environment, where WS2@COF exhibits a more negative adsorption energy for H2O2, effectively enriching H2O2 in the catalytic edge sites of WS2. Furthermore, the internal electric field at the WS2 and COF interface accelerates electron flow, boosting WS2's catalytic activity and achieving domain‐limited catalytic enhancement of ECL. Self‐designed DNA nanomachines combined with cascading molecular keypad locking mechanisms are integrated into the biosensors, effectively guaranteeing the accuracy of the sensing process while providing crucial safeguards for molecular diagnostics and information security applications. In essence, this innovative approach represents the first system to enhance local free radical concentrations by enriching co‐reactants on the electrode surface through nanoconfinement catalysis, yielding heightened ECL luminescence intensity. The potential impact of this novel strategy and sensing mechanism on real‐bioanalysis applications is promising.

Mapping Endocytic Vesicular Acidification with a pH-Responsive DNA Nanomachine.

Mapping the endocytic vesicular acidification process is of prior importance to better understand the health and pathological processes of cells. Herein, by integrating a pH-sensitive i-motif and a pair of fluorescence resonance energy transfer (FRET) into a tetrahedral DNA framework (TDF), we develop a pH-responsive DNA nanomachine, allowing for efficient sensing of pH from 7.0 to 5.5 via the pH-triggered spatial proximity modulation of FRET. The inheriting endo-lysosome-targeting ability of TDF enables spatiotemporal tracking of endocytic vesicle acidification during the endosomal maturation process. Analysis of pH-dependent FRET response at single fluorescent spot level reveals the significant difference of endocytic vesicular acidification between normal and cancer cells. The performance of pH-responsive DNA nanomachine underlines its potential for studies on vesicle acidification-related pathologies as a universal platform.

Endogenous Messenger RNA-Stimulative Self-Enhanced Orthogonal Catalytic DNA Nanomachine under Near-Infrared Light Mediation for High-Performance Imaging in Living Biosamples

Endogenous Messenger RNA-Stimulative Self-Enhanced Orthogonal Catalytic DNA Nanomachine under Near-Infrared Light Mediation for High-Performance Imaging in Living Biosamples

DNA nanomachine‐driven chemodynamic therapy against glioblastoma

AbstractChemodynamic therapy (CDT) has shown promising antitumor effects in various malignant tumors. However, its application for glioblastoma (GBM) is significantly hindered by the challenge of delivering CDT agents across the blood‐brain barrier (BBB) and achieving efficient tumor targeting. To overcome these obstacles, this study presents a novel DNA nanomachine (Cu@tFNAs‐G‐A NM) by loading copper ions (Cu2+) onto tetrahedral framework nucleic acids (tFNAs) functionalized with dual DNA aptamers. The dual DNA aptamers (GS24 for BBB penetration and AS1411 for tumor targeting) empowered Cu@tFNAs‐G‐A NM with the ability to effectively penetrate the BBB and selectively accumulate in tumor cells. Upon internalization, the loaded Cu2+ reacted with tumor‐overexpressed reductive glutathione (GSH) and hydrogen peroxide (H2O2), generating hydroxyl radicals (·OH) and inducing tumor cell death. Additionally, Cu@tFNAs‐G‐A NM was found to be rapidly cleared from the brain and normal tissues within 24 h, minimizing potential systemic toxic side effects. These findings demonstrate the promising potential of Cu@tFNAs‐G‐A NM for effective CDT against GBM and open up new avenues for the development of targeted therapies for GBM.

Label-free and ultrasensitive detection of environmental lead ions based on spatially localized DNA nanomachines driven by hyperbranched hybridization chain reaction

A label-free fluorescent sensing strategy for the rapid and highly sensitive detection of Pb2+ was developed by integrating Pb2+ DNAzyme-specific cleavage activity and a tetrahedral DNA nanostructure (TDN)-enhanced hyperbranched hybridization chain reaction (hHCR). This strategy provides accelerated reaction rates because of the highly effective collision probability and enriched local concentrations from the spatial confinement of the TDN, thus showing a higher detection sensitivity and a more rapid detection process. Moreover, a hairpin probe based on a G-triplex instead of a G-quadruplex or chemical modification makes hybridization chain reaction more controlled and flexible, greatly improving signal amplification capacities and eliminating labeled DNA probes. The enhanced reaction rates and improved signal amplification efficiency endowed the biosensors with high sensitivity and a rapid response. The label-free detection of Pb2+ based on G-triplex combined with thioflavin T can be achieved with a detection limit as low as 1.8 pM in 25 min. The proposed Pb2+-sensing platform was also demonstrated to be applicable for Pb2+ detection in tap water, river water, shrimp, rice, and soil samples, thus showing great potential for food safety and environmental monitoring.

Continuous input DNA Nanomachine to program automated tumor diagnosis and treatment system

The diagnosis of a single disease marker can provide important information about the occurrence and progression of the disease, but a single marker may not be sufficient to identify a certain type of disease and lead to false-positive results. Molecular logic gates provide smart options for simultaneous detection of biomarkers. In order to diagnose early-stage cancer with high sensitivity and directly perform automated precision treatment on identified cancer cells. In this study, all identification and computation modules were integrated into a single structure to construct a Pd@Pt ONPs-DNA nanomachine logic gate nanodevice, which enables specific recognition of three overexpressed cancer cell biomarkers. When any of the biomarkers are overexpressed, the OR logic circuit outputs a fluorescent signal, indicating the occurrence of a positive event. At the same time, the resulting fluorescence signal is then fed directly into the AND logic gate, when the input is (1, 1, 1), a laser output of 808 nm is generated. The photothermal properties and nanozyme activity of Pd@Pt ONPs convert the 808 nm laser into cytotoxicity, using continuously connected OR and AND gates for early warning of cancer and automatic and precise treatment.

Photocurrent switchable dual-target bioassay: Signal distinction and interface reconfiguration via pH-responsive triplex DNA programming

Most multiplexed photoelectrochemical (PEC) sensors require additional instrumentation and cumbersome electrode modification and surface partitioning, which limits their portability and instrument miniaturization. Herein, a pH-responsive programmable triple DNA nanomachine was developed for constructing a reconfigurable multiplex PEC sensing platform. By programming the base sequence, T-A·T-riched triple DNA was designed to construct integrated nano-controlled release machine (INCRM) for simultaneous recognition of multiple targets. The INCRM enables to recognize two targets in one step, and sequentially separate the signal labels by pH adjustment. The detached signal label catalyzes glucose to produce gluconic acid, causing the C-riched DNA fold into a triple structure on the electrode surface. As a result, one target can be detected relying on the enhanced photocurrent due to accelerated electron transfer between the CdS QD labeled at the end of C-riched DNA and the electrode. The triplex DNA dissociation in pH 7.4 buffer reconfigures the electrode interface, which can be continued to detect another target. The feasibility of the multiplexed sensor is verified by the detection of extensively coexisting antibiotics enrofloxacin (ENR) and ciprofloxacin (CIP). Under the optimal conditions, wide linear range (10 fg/mL ∼ 1 μg/mL) and low detection limit (3.27 fg/mL and 9.60 fg/mL) were obtained. The pH-regulated programmable triplex DNA nanomachine-based sensing platform overcomes the technical difficulties of conventional multiplexed PEC assay, which may open the way for miniaturization of multiplexed PEC sensors.

Quantitative Chemical Imaging of Organelles.

ConspectusDNA nanodevices are nanoscale assemblies, formed from a collection of synthetic DNA strands, that may perform artificial functions. The pioneering developments of a DNA cube by Nadrian Seeman in 1991 and a DNA nanomachine by Turberfield and Yurke in 2000 spawned an entire generation of DNA nanodevices ranging from minimalist to rococo architectures. Since our first demonstration in 2009 that a DNA nanodevice can function autonomously inside a living cell, it became clear that this molecular scaffold was well-placed to probe living systems. Its water solubility, biocompatibility, and engineerability to yield molecularly identical assemblies predisposed it to probe and program biology.Since DNA is a modular scaffold, one can integrate independent or interdependent functionalities onto a single assembly. Work from our group has established a new class of organelle-targeted, DNA-based fluorescent reporters. These reporters comprise three to four oligonucleotides that each display a specific motif or module with a specific function. Given the 1:1 stoichiometry of Watson-Crick-Franklin base pairing, all modules are present in a fixed ratio in every DNA nanodevice. These modules include an ion-sensitive dye or a detection module and a normalizing dye for ratiometry that along with detection module forms a "measuring module". The third module is an organelle-targeting module that engages a cognate protein so that the whole assembly is trafficked to the lumen of a target organelle. Together, these modules allow us to measure free ion concentrations with accuracies that were previously unattainable, in subcellular locations that were previously inaccessible, and at single organelle resolution. By revealing that organelles exist in different chemical states, DNA nanodevices are providing new insights into organelle biology. Further, the ability to deliver molecules with cell-type and organelle level precision in animal models is leading to biomedical applications.This Account outlines the development of DNA nanodevices as fluorescent reporters for chemically mapping or modulating organelle function in real time in living systems. We discuss the technical challenges of measuring ions within endomembrane organelles and show how the unique properties of DNA nanodevices enable organelle targeting and chemical mapping. Starting from the pioneering finding that an autonomous DNA nanodevice could map endolysosomal pH in cells, we chart the development of strategies to target organelles beyond the endolysosomal pathway and expanding chemical maps to include all the major ions in physiology, reactive species, enzyme activity, and voltage. We present a series of vignettes highlighting the new biology unlocked with each development, from the discovery of chemical heterogeneity in lysosomes to identifying the first protein importer of Ca2+ into lysosomes. Finally, we discuss the broader applicability of targeting DNA nanodevices organelle-specifically beyond just reporting ions, namely using DNA nanodevices to modulate organelle state, and thereby cell state, with potential therapeutic applications.

Programmable DNA Nanomachine Integrated with Electrochemically Controlled Atom Transfer Radical Polymerization for Antibody Detection at Picomolar Level.

The quantitative detection of antibodies is crucial for the diagnosis of infectious and autoimmune diseases, while the traditional methods experience high background signal noise and restricted signal gain. In this work, we have developed a highly efficient electrochemical biosensor by constructing a programmable DNA nanomachine integrated with electrochemically controlled atom transfer radical polymerization (eATRP). The sensor works by binding the target antidigoxin antibody (anti-Dig) to the epitope of the recognization probe, which then initiates the cascaded strand displacement reaction on a magnetic bead, leading to the capture of cupric oxide (CuO) nanoparticles through magnetic separation. After CuO was dissolved, the eATRP initiators were attached to the electrode based on the CuΙ-catalyzed azide-alkyne cycloaddition. The subsequent eATRP reaction results in the formation of long electroactive polymers (poly-FcMMA), producing an amplified current response for sensitive detection of anti-Dig. This method achieved a detection limit at clinically relevant picomolar concentration in human serum, offering a sensitive, convenient, and cost-effective tool for detecting various biomarkers in a wide range of applications.

Cleaving Folded RNA by Multifunctional DNAzyme Nanomachines.

Both tight and specific binding of folded biological mRNA is required for gene silencing by oligonucleotide gene therapy agents. However, this is fundamentally impossible using the conventional oligonucleotide probes according to the affinity/specificity dilemma. This study addresses this problem for cleaving folded RNA by using multicomponent agents (dubbed 'DNA nanomachine' or DNM). DNMs bind RNA by four short RNA binding arms, which ensure tight and highly selective RNA binding. Along with the improved affinity, DNM maintain the high sequence selectivity of the conventional DNAzymes. DNM enabled up to 3-fold improvement in DNAzymes catalytic efficiency (kcat/Km) by facilitating both RNA substrate binding and product release steps of the catalytic cycle. This study demonstrates that multicomponent probes organized in sophisticated structures can help to achieve the balance between affinity and selectivity in recognizing folded RNA and thus creates a foundation for applying complex DNA nanostructures derived by DNA nanotechnology in gene therapy.

A highly cost-effective and sensitive biosensing platform: Fuel-propelled DNA nanomachines modulate fluorescence switching of DNA silver nanoclusters

It is of great significance to develop a low-cost, simple but highly sensitive fluorescence sensing platform for low-abundance molecule detection. However, traditional organic fluorophore labeling often requires expensive and sophisticated synthesis, making it difficult to use in resource-limited areas. Besides, the traditional “one-to-one” detection mechanism presents an obstacle to sensitive detection of low-level molecules, because one target only triggers one signal. To tackle these predicaments, in this work, a brightly red-emissive three-stranded DNA stabilized silver nanocluster (Ag NC) has been successfully synthesized. However, the fluorescence emission of Ag NCs can be darkened when it is in a loose single-stranded DNA. Building on this finding, we elaborately engineer a highly cost-effective and sensitive biosensing platform, which exploits a target-triggered and fuel-driven DNA nanomachine to precisely manipulate the structural transformation of Ag NCs from the rigid three-stranded DNA structures to the flexible single-stranded DNA, accordingly achieving the fluorescence switching for amplified sensing of low-abundance molecules. It is worth noting that nucleases and complex secondary structures are not involved in the entire system, offering a simple and flexible design. Furthermore, the novel and red-emissive DNA Ag NCs are employed as signal reporting, avoiding expensive and time-consuming organic fluorophore labeling. Experiment results demonstrate that this newly developed biosensing platform has a high signal-to-noise (∼15-folds), low limit of detection (1.59 pM), and good specificity to H5N1 DNA. Importantly, it can be used for H5N1 DNA detection in complex biological samples with a satisfactory recovery.

Bimodal 3D DNA nanomachines coupling with bioconjugates powered electrochemical and visual-driven dual-enzyme cascade sensing of Let-7a

A dual-enzyme self-powered biosensor utilizing bimodal 3D DNA nanomachines and gold nanoparticle (AuNPs)/carbon/disulfide molybdenum hollow nanorods bioconjugates is developed for the electrochemical and visual detection of the tumor biomarker let-7a. DNA probes are immobilized on a flexible electrode substrate of AuNPs/graphdiyne/carbon cloth to improve probe loading capacity and electron transfer efficiency. The presence of let-7a initiates the work of dual-enzyme 3D DNA nanomachine, and the output DNA single strand cleaved by Nb.BbvCI is served as a bridge to connect glucose oxidase (GOD)-DNA conjugate. The GOD coordinates with the cathodic enzyme bilirubin oxidase and produces an increased open circuit voltage signal, which is attributed to the hexaammine ruthenium in the double-stranded adsorption system of the biocathode. By utilizing the H2O2 generated during the oxidation process of GOD, a linear relationship between the color of 3,3′,5,5′-tetramethylbenzidine and the concentration of let-7a is established, enhancing the reliability of the detection results through the independent signal conversion mechanisms of electrochemical/colorimetric modes. The results demonstrate a wide dynamic range of 0.0001–0.1 pM with detection limits of 31.04 aM (S/N=3) in electrochemical mode and 59.99 aM (S/N=3) in the colorimetric mode. This dual-mode biosensing strategy can be expanded to detect additional nucleic acid biomarkers, providing a promising support and reference for biological analysis and medical diagnosis.

Endogenous AND Logic DNA Nanomachine for Highly Specific Cancer Cell Imaging.

Intracellular cancer-related biomarker imaging strategy has been used for specific identification of cancer cells, which was of great importance to accurate cancer clinical diagnosis and prognosis studies. Localized DNA circuits with improved sensitivity showed great potential for intracellular biomarkers imaging. However, the ability of localized DNA circuits to specifically image cancer cells is limited by off-site signal leakage associated with a single-biomarker sensing strategy. Herein, we integrated the endogenous enzyme-powered strategy with logic-responsive and localized signal amplifying capability to construct a self-assembled endogenously AND logic DNA nanomachine (EDN) for highly specific cancer cell imaging. When the EDN encountered a cancer cell, the overexpressed DNA repairing enzyme apurinic/apyrimidinic endonuclease 1 (APE1) and miR-21 could synergistically activate a DNA circuit via cascaded localized toehold-mediated strand displacement (TMSD) reactions, resulting in amplified fluorescence resonance energy transfer (FRET) signal. In this strategy, both endogenous APE1 and miR-21, served as two "keys" to activate the AND logic operation in cancer cells to reduce off-tumor signal leakage. Such a multiplied molecular recognition/activation nanomachine as a powerful toolbox realized specific capture and reliable imaging of biomolecules in living cancer cells.

Sensing multiplex microRNAs using dual-functional fork-type-track DNA nanomachine on optical fiber

We propose a simple and robust biosensing platform which can be used for accurate determination of multiplex microRNA targets in biological samples. We develop the sensor by immobilizing a novel DNA nanomachine system on the surface of a gold-nanoparticles-modified optical fiber, which contains a uniquely designed dual-functional fork-type track chain with a FAM-labeled DNAzyme recognition site on one branch and a rolling-loop-amplification (RCA) primer on the other. The strategy allows two amplification strategies, i.e., DNA walking and RCA, to be easily achieved as the target microRNAs are in the sample to initiate the corresponding reactions. Two different microRNAs (miRNA-21 and miRNA-375 in the study) are independently and accurately determined without interference from each other, by respectively measuring fluorescence and chemiluminescence signals generated from DNA walking and RCA. The results show that the method is capable for multiplex micorRNA assays with high sensitivity (limit-of-detection 0.21 pM for miRNA-21 and 0.018 pM for miRNA-375) and high selectivity, and the sensor exhibits good repeatability and storage stability. The method successfully determines microRNAs in MCF-7, Hela and MCF-10A cells, showing its ability to analyze real complex biological samples.

Target-induced DNA nanomachine operation for the detection of proteins

Accurate and sensitive detection of disease-associated proteins in early stage of patients plays an important role in timely treatment and successfully extending patients' lives. To meet this demand, we herein rationally designed a flexible target-induced DNA nanomachine operation (TIDNMO) sensor for the detection of proteins. The TIDNMO system was composed of DNA nanoswitch and DNA walker. Triplex DNA nanoswitch was triggered by specific target, followed by the release of the walking strand, which initiated the DNA walker amplification as signal output. In addition, the Exo III could drive walking strand autonomously move on gold nanoparticle surface to realize 2 orders of magnitude signal amplification. What's more, this sensor could transform its suitable functional recognition element of DNA nanoswitch to recognize other specific molecule and realize different targets sensing based on identical walking tracks. Considering the facile reporter elements and efficient amplification performance, the present DNA nanomachine as a sensor could achieve a detection limit of 68 pM for anti-Dig antibody, 0.95 pM for mucin-1 respectively, along with a superb specificity. Furthermore, the method reported here opened a new chapter in disease-related protein sensing for the development of clinical early diagnosis.

Homogeneous electrochemical ratiometric biosensor for MircoRNA detection based on UiO-66-NH2 signal probe and waste-free entropy-driven DNA machine

The construction of efficient methods for highly sensitive and rapid detection of disease markers is essential for the early diagnosis of serious diseases. In this paper, taking advantage of the UiO-66-NH2 signal molecule in combination with a waste-free entropy-driven DNA machine, a novel homogeneous electrochemical ratiometric platform is developed to detect MircoRNA (miRNA). Metal-organic framework materials (UiO-66-NH2 MOF) and ferrocene were utilized as electrochemical signal tags and reference probes, respectively. The target-initiated waste-free three-dimensional (3D) entropy-driven DNA nanomachine is activated in the presence of miRNA, resulting in DNA-labeled-UiO-66-NH2 falling off from the electrode, leading to a decrease in the signal of UiO-66-NH2 at 0.83V. Our strategy can mitigate false positive responses induced by the DNA probes immobilized on electrodes in traditional distance-dependent signal adjustment ratiometric strategies. The proposed ratiometric platform demonstrates superior sensitivity (a detection limit of 9.8 fM), simplified operation, high selectivity, and high repeatability. The ratiometric biosensor is also applied to detect miRNA content in spiked serum samples.

Ultrasensitive serological digoxin fluorescence sensor based on cascade amplification of entropy-driven DNA nanomachine and proximity ligation assay

A nanobiosensor was prepared by DNA nanomachine based on proximity ligation assay (PLA) for the detection of digoxin (DIG) in serum. In this sensor the DNA nanomachine releases a large number of signal probes (SP) after addition of DIG, which participate in PLA as the connector strand, initiating real-time quantitative polymerase chain reaction (qPCR) and producing fluorescence signals. For the benefit of clinical therapeutics, we determined the detection range of 0.1 ∼ 5 ng/mL. Under the optimal experimental conditions, the cycle threshold (Ct) value showed a good linear relationship with DIG concentrations, and the detection limit was as low as 0.06 ng/mL (3σ). This method achieved the ultrasensitive detection of DIG by signal amplification and was successfully used for the detection of DIG in real serum, which has a good potential for application in the field of therapeutic drug monitoring.

Amplified and Specific Staining of Protein Dimerization on Cell Membrane Catalyzed by Responsively Installed DNA Nanomachines for Cancer Diagnosis (Adv. Healthcare Mater. 10/2024)

Amplified and Specific Staining of Protein Dimerization on Cell Membrane Catalyzed by Responsively Installed DNA Nanomachines for Cancer Diagnosis (Adv. Healthcare Mater. 10/2024)