DNA Hybridization Research Articles

DNA Origami‐Directed Self‐Assembly of Gold Nanospheres for Plasmonic Metasurfaces

AbstractPlasmonic nanostructures are frequently utilized to create metasurfaces with a large variety of optical effects. Control over shape and positioning of the nanostructures is key to the function of such plasmonic metasurfaces. Next to lithographic means, directed self‐assembly is a viable route to create plasmonic structures on surfaces with the necessary precision. Here, a combined approach of DNA origami self‐assembly and electron beam lithography is presented for determinate positioning of gold nanospheres on a SiO2 surface. First, DNA origami structures bind to the electron beam‐patterned substrate and subsequently, gold nanoparticles attach to a defined binding site on the DNA origami structure via DNA hybridization. A sol‐gel reaction is then used to grow a silica layer around the DNA, thereby increasing the stability of the self‐assembled metasurface. A mean yield of 74% of single gold nanospheres is achieved located at the determinate positions with a spatial position accuracy of 9 nm. Gold nanosphere dimers and trimers are achieved with a rate of 65% and 60%, respectively. The applicability of this structuring method is demonstrated by the fabrication of metasurfaces whose optical response can be tuned by the polarization of the incoming and the scattered light.

Machine learning based DNA melt curve profiling enables automated novel genotype detection

Surveillance for genetic variation of microbial pathogens, both within and among species, plays an important role in informing research, diagnostic, prevention, and treatment activities for disease control. However, large-scale systematic screening for novel genotypes remains challenging in part due to technological limitations. Towards addressing this challenge, we present an advancement in universal microbial high resolution melting (HRM) analysis that is capable of accomplishing both known genotype identification and novel genotype detection. Specifically, this novel surveillance functionality is achieved through time-series modeling of sequence-defined HRM curves, which is uniquely enabled by the large-scale melt curve datasets generated using our high-throughput digital HRM platform. Taking the detection of bacterial genotypes as a model application, we demonstrate that our algorithms accomplish an overall classification accuracy over 99.7% and perform novelty detection with a sensitivity of 0.96, specificity of 0.96 and Youden index of 0.92. Since HRM-based DNA profiling is an inexpensive and rapid technique, our results add support for the feasibility of its use in surveillance applications.

Discovery and characterization of a novel pathogen Erwinia pyri sp. nov. associated with pear dieback: taxonomic insights and genomic analysis

In 2022, a novel disease similar to pear fire blight was found in a pear orchard in Zhangye City, Gansu Province, China. The disease mainly damages the branches, leaves, and fruits of the plant. To identify the pathogen, tissue isolation and pathogenicity testing (inoculating the potential pathogen on healthy plant tissues) were conducted. Furthermore, a comprehensive analysis encompassing the pathogen’s morphological, physiological, and biochemical characteristics and whole-genome sequencing was conducted. The results showed that among the eight isolates, the symptoms on the detached leaves and fruits inoculated with isolate DE2 were identical to those observed in the field. Verifying Koch’s postulates confirmed that DE2 was the pathogenic bacterium that causes the disease. Based on a 16S rRNA phylogenetic tree, isolate DE2 belongs to the genus Erwinia. Biolog and API 20E results also indicated that isolate DE2 is an undescribed species of Erwinia. Isolate DE2 was negative for oxidase. Subsequently, the complete genome sequence of isolate DE2 was determined and compared to the complete genome sequences of 29 other Erwinia species based on digital DNA–DNA hybridization (dDDH) and average nucleotide identity (ANI) analyses. The ANI and dDDH values between strain DE2 and Erwinia species were both below the species thresholds (ANI < 95–96%, dDDH<70%), suggesting that isolate DE2 is a new species of Erwinia. We will temporarily name strain DE2 as Erwinia pyri sp. nov. There were 548 predicted virulence factors in the genome of strain DE2, comprising 534 on the chromosome and 5 in the plasmids. The whole genome sequence of strain DE2 has been submitted to the NCBI database (ASM3075845v1) with accession number GCA_030758455.1. The strain DE2 has been preserved at the China Center for Type Culture Collection (CCTCC) under the deposit number CCTCC AB 2024080. This study represents the initial report of a potentially new bacterial species in the genus Erwinia that causes a novel pear dieback disease. The findings provide a valuable strain resource for the study of the genus Erwinia and establish a robust theoretical foundation for the prevention and control of emerging pear dieback diseases.

Two Novel Alkaliphilic Species Isolated from Saline-Alkali Soil in China: Halalkalibacter flavus sp. nov., and Halalkalibacter lacteus sp. nov

The degradation of farmland in China underscores the need for developing and utilizing saline-alkali soil. Soil health relies on microbial activity, which aids in the restoration of the land’s ecosystem, and hence it is important to understand microbial diversity. In the present study, two Gram-stain-positive strains HR 1-10T and J-A-003T were isolated from saline-alkali soil. Preliminary analysis suggested that these strains could be a novel species. Therefore, the taxonomic positions of these strains were evaluated using polyphasic analysis. Phylogenetic and 16S rRNA gene sequence analysis indicated that these strains should be assigned to the genus Halalkalibacter. Cell wall contained meso-2,6-diaminopimelic acid. The polar lipids present in both strains were diphosphatidyl-glycerol, phosphatidylglycerol, and an unidentified phospholipid. The major fatty acids (>10%) were anteiso-C15:0, C16:0 and iso-C15:0. Average nucleotide identity and digital DNA#x2013;DNA hybridization values were below the threshold values (95% and 70%, respectively) for species delineation. Based on the above results, the strains represent two novel species of the genus Halalkalibacter, for which the names Halalkalibacter flavus sp. nov., and Halalkalibacter lacteus sp. nov., are proposed. The type strains are HR 1-10T (=GDMCC 1.2946T = MCCC 1K08312T = JCM 36285T), and J-A-003T (=GDMCC 1.2949T = MCCC 1K08417T = JCM 36286T).

DNA-Based Molecular Machines: Controlling Mechanisms and Biosensing Applications

The rise of DNA nanotechnology has driven the development of DNA-based molecular machines, which are capable of performing specific operations and tasks at the nanoscale. Benefitting from the programmability of DNA molecules and the predictability of DNA hybridization and strand displacement, DNA-based molecular machines can be designed with various structures and dynamic behaviors and have been implemented for wide applications in the field of biosensing due to their unique advantages. This review summarizes the reported controlling mechanisms of DNA-based molecular machines and introduces biosensing applications of DNA-based molecular machines in amplified detection, multiplex detection, real-time monitoring, spatial recognition detection, and single-molecule detection of biomarkers. The challenges and future directions of DNA-based molecular machines in biosensing are also discussed.

On the thermodynamics of DNA double strand in the Peyrard-Bishop-Dauxois model

The temperature dependence of energy, partition function, entropy and free energy, describing a first-order-like phase transition of a DNA molecule is calculated based on the direct method of molecular dynamics for the classical Peyrard-Bishop-Dauxois model. It is shown that without taking into account the quantum freezing of the degrees of freedom at low temperatures, the relationship between these quantities is not determined. The latent heat of DNA melting is calculated for homogeneous PolyA/PolyT and PolyG/PolyC DNA chains. It is shown that this melting transition corresponds to a sharp temperature dependence of the heat capacity.

Bimetallic DNAsome Decorated with G4-DNA as a Nanozyme for Targeted and Enhanced Chemo/Chemodynamic Cancer Therapy.

Cancer is indisputably one of the major threats to mankind, and hence the design of new approaches for the improvement of existing therapeutic strategies is always wanted. Herein, the design of a tumor microenvironment-responsive, DNA-based chemodynamic therapy (CDT) nanoagent with dual Fenton reaction centers for targeted cancer therapy is reported. Self-assembly of DNA amphiphile containing copper complex as the hydrophobic Fenton reaction center results in the formation of CDT-active DNAsome with Cu2+-based Fenton catalytic site as the hydrophobic core and hydrophilic ssDNA protrude on the surface. DNA-based surface addressability of the DNAsome is then used for the integration of second Fenton reaction center, which is a peroxidase-mimicking DNAzyme noncovalently loaded with Hemin and Doxorubicin, via DNA hybridization to give a CDT agent having dual Fenton reaction centers. Targeted internalization of the CDT nanoagent and selective generation of •OH inside HeLa cell are also shown. Excellent therapeutic efficiency is observed for the CDT nanoagent both in vitro and in vivo, and the enhanced efficacy is attributed to the combined and synergetic action of CDT and chemotherapy.

A novel signal amplified sandwich aptamer-based visual lateral flow assay based on self-assembly bifunctional nucleic acid for rapid and sensitive detection of CKMB

At present, rapid and sensitive detection of disease biomarkers is still the focus of early clinical diagnosis. As a rapid response to point-of-care testing (POCT), lateral flow assay (LFA) has been given serious attention. Unfortunately, due to the sensitivity limitations, LFA, especially AuNPs-based LFA, was difficult to play an important role in the detection of trace biomarkers. The combination of different signal amplification methods increases the complexity of operation and use, or prolongs the detection time, which sacrifices the advantages of fast and convenient of LFA. Hence, a novel sandwich aptamer-based visual LFA based on bifunctional nucleic acid (bFNA-LFA) was proposed for rapid and sensitive detection of creatine kinase MB (CKMB) in this study. Based on hybridization chain reaction (HCR) and nucleic acid hybridization, a bifunctional nucleic acid with target recognition and signal amplification was prepared, which effectively improved the sensitivity of CKMB detection without increasing the reaction time and depending on the detecting equipment. Under optimal reaction conditions, the limit of detection of 7.92 ng/mL was observed, which was 20 times lower than that of sandwich aptamer-based AuNPs LFA in the previous study. The proposed bFNA-LFA assay expanded a new idea in improving the sensitivity of LFA, and provided a reference for the subsequent application of nucleic acid nanomaterials in the detection of protein biomarkers.

Linker histone H1 regulates homeostasis of heterochromatin-associated cRNAs

Chromatin-associated RNAs (cRNAs) are a poorly characterized fraction of cellular RNAs that co-purify with chromatin. Their full complexity and the mechanisms regulating their packaging and chromatin association remain poorly understood. Here, we address these questions in Drosophila. We find that cRNAs constitute a heterogeneous group of RNA species that is abundant in heterochromatic transcripts. We show that heterochromatic cRNAs interact with the heterogeneous nuclear ribonucleoproteins (hnRNP) hrp36/hrp48 and that depletion of linker histone dH1 impairs this interaction. dH1 depletion induces the accumulation of RNA::DNA hybrids (R-loops) in heterochromatin and, as a consequence, increases retention of heterochromatic cRNAs. These effects correlate with increased RNA polymerase II (RNAPII) occupancy at heterochromatin. Notably, impairing cRNA assembly by depletion of hrp36/hrp48 mimics heterochromatic R-loop accumulation induced by dH1 depletion. We also show that dH1 depletion alters nucleosome organization, increasing accessibility of heterochromatin. Altogether, these perturbations facilitate annealing of cRNAs to the DNA template, enhancing R-loop formation and cRNA retention at heterochromatin.

Characterizing the Loss and Acquisition of Tn2-like Transposon in Klebsiella pneumoniae: Implications for Carbapemase Gene Dissemination

Over one year, two KPC-producing and two non-KPC-producing Klebsiella pneumoniae strains were isolated from a patient. Genome and DNA hybridization analyses revealed the first three strains as a clonal lineage, with carbapenem resistance changes due to a Tn2-like transposon on an IncR/IncFII plasmid. The fourth strain, carrying three plasmids, caused a lethal infection and represented a different lineage. All strains belonged to the ST11-SL47-OL101 type. This study highlights the Tn2-like transposon's role in carbapenemase gene spread and the importance of distinguishing between bacterial colonization and infection.

Simulation-Guided Rational Design of DNA Walker-Based Theranostic Platform.

Biomolecule-functionalized nanoparticles represent a type of promising biomaterials in biomedical applications owing to their excellent biocompatibility and versatility. DNA-based reactions on nanoparticles have enabled emerging applications including intelligent biosensors, drug delivery, and biomimetic devices. Among the reactions, strand hybridization is the critical step to control the sensitivity and specificity of biosensing, and the efficiency of drug delivery. However, a comprehensive understanding of DNA hybridization on nanoparticles is still lacking, which may differ from the process in homogeneous solutions. To address this limitation, coarse-grained model-based molecular dynamic simulation is harnessed to disclose the critical factors involved in intermolecular hybridization. Based on simulation guidance, DNA walker-based smart theranostic platform (DWTP) based on "on-particle" hybridization is developed, showing excellent consistency with simulation. DWTP is successfully applied for highly sensitive miRNA 21 detection and tumor-specific miRNA 21 imaging, driven by tumor-endogenous APE 1 enzyme. It enables the precise release of antisense oligonucleotide triggered by tumor-endogenous dual-switch miRNA 21 and APE 1, facilitating effective gene silencing therapy with high biosafety. The simulation of "on-particle" DNA hybridization has improved the corresponding biosensing performance and the release efficiency of therapeutic agents, representing a conceptually new approach for DNA-based device design.

Impact of Pericoronary Microbiota Composition on Course of Recovery after Third Molar Alveotomy

Although the role of microbiota has been investigated in relation to different oral diseases, it is unknown if its composition has any effect on the course of recovery after third molar alveotomy. Our aim was to determine the influence of patient clinical characteristics as well as pericoronary microbiota composition on the course of recovery after a semi-impacted third molar alveotomy. Thirty-six patients were included and samples obtained with paper points, swabs, and tissue samples were analyzed using DNA hybridization and culture methods. Among the 295 organisms detected, the most frequent were Streptococcus spp. (22.4%; 66/295) followed by Fusobacterium spp. (11.9%; 35/295), and T. forsythia (9.1%; 27/295). A comparison of microbiota composition in patients with better and worse recovery did not show significant differences. Worse recovery outcomes were more frequent in patients with a grade 2 self-assessment of oral health (p = 0.040) and better recovery courses were observed in patients with a grade 4 self-assessment (p = 0.0200). A worse recovery course was statistically significant more frequently in patients with previous oral surgical procedures (p = 0.019). Although we demonstrate that worse recovery outcomes were more frequent when certain bacteria were detected, there was no statistically significant difference. Further research is needed to identify microbial profiles specific to the development of worse outcomes after a third molar alveotomy.

Development of supramolecular anticoagulants with on-demand reversibility.

Drugs are administered at a dosing schedule set by their therapeutic index, and termination of action is achieved by clearance and metabolism of the drug. In some cases, such as anticoagulant drugs or immunotherapeutics, it is important to be able to quickly reverse the drug's action. Here, we report a general strategy to achieve on-demand reversibility by designing a supramolecular drug (a noncovalent assembly of two cooperatively interacting drug fragments held together by transient hybridization of peptide nucleic acid (PNA)) that can be reversed with a PNA antidote that outcompetes the hybridization between the fragments. We demonstrate the approach with thrombin-inhibiting anticoagulants, creating very potent and reversible bivalent direct thrombin inhibitors (Ki = 74 pM). The supramolecular inhibitor effectively inhibited thrombus formation in mice in a needle injury thrombosis model, and this activity could be reversed by administration of the PNA antidote. This design is applicable to therapeutic targets where two binding sites can be identified.

Genome-based analysis of the family Paracoccaceae and description of Ostreiculturibacter nitratireducens gen. nov., sp. nov., isolated from an oyster farm on a tidal flat

Two bacterial strains, designated FR2A1T and MT2-5-38, were isolated from the surface sediments of an oyster farm on a tidal flat in Quanzhou Bay, China. Both strains were Gram-stain-negative, rod-shaped, aerobic, catalase-positive, and oxidase-positive. The 16S rRNA gene sequences of the two strains were 100% identical and had the highest similarity (97.1%) with Phaeovulum vinaykumarii JA123T. The average nucleotide identity (ANI) value and digital DNA–DNA hybridization (DDH) value indicated that the two strains belonged to a single species. Gene annotation revealed that the two strains contained a gene cluster for nitrate reduction and a gene cluster for sulfur oxidation, indicating a possible role in N and S cycling in the tidal flat sediment. The phylogeny inferred from the 16S rRNA gene and 120 conserved proteins indicated that the two strains formed a distinct monophyletic clade within the family Paracoccaceae. The respiratory quinone was Q-10. The major fatty acids consisted of summed feature 8 (C18:1ω7c and/or C18:1ω6c) and C18:0. The polar lipids consisted of phosphatidylethanolamine, phosphatidylglycerol, and several unidentified phospholipids. Based on the above characteristics, strains FR2A1T and MT2-5-38 represent a novel genus and a novel species, for which we propose the name Ostreiculturibacter nitratireducens gen. nov., sp. nov. The type strain is FR2A1T (=MCCC 1K08809T = KCTC 8317T). Phylogenomic analysis of 1,606 high-quality genomes of the family Paracoccaceae, including type strains, non-type strains, and uncultivated bacteria, was performed using the Genome Taxonomic Database Toolkit (GTDB-Tk), and the average amino acid identity (AAI) value of the phylogenetic clade was estimated. We found that 35 species of the family Paracoccaceae needed re-classification, and an AAI value of 70% was chosen as the genus boundary within the family Paracoccaceae.

Oerskovia flava sp. nov., a deoxynivalenol (DON)-degrading actinomycete isolated from the rhizosphere soil of long-term continuous cropping cucumber.

The deoxynivalenol (DON)-degrading bacterium JB1-3-2T was isolated from a rhizosphere soil sample of cucumber collected from a greenhouse located in Zhenjiang, Eastern China. The JB1-3-2T strain is a Gram-stain-positive, nonmotile and round actinomycete. Growth was observed at temperatures between 15 and 40 ℃ (optimum, 35 ℃), in the presence of 15% (w/v) NaCl (optimum, 3%), and at pH 3 and 11 (optimum, 7). The major cellular fatty acids identified were anteiso-C15:0, iso-C16:0 and anteiso-C17:0. Genome sequencing revealed a genome size of 4.11Mb and a DNA G + C content of 72.5mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the JB1-3-2T strain was most closely related to type strains of the Oerskovia species, with the highest sequence similarity to Oerskovia turbata NRRL B-8019T (98.2%), and shared 98.1% sequence identity with other valid type strains of this genus. Digital DNA‒DNA hybridization (dDDH) and average nucleotide identity (ANI) showed 21.8-22.2% and 77.2-77.3% relatedness, respectively, between JB1-3-2T and type strains of the genus Oerskovia. Based on genotypic, phylogenetic, chemotaxonomic, physiological and biochemical characterization, Oerskovia flava, a novel species in the genus Oerskovia, was proposed, and the type strain was JB1-3-2T (= CGMCC 1.18555T = JCM 35248T). Additionally, this novel strain has a DON degradation ability that other species in the genus Oerskovia do not possess, and glutathione-S-transferase was speculated to be the key enzyme for strain JB1-3-2T to degrade DON.

Structure-based drug design of DNA minor groove binders and evaluation of their antibacterial and anticancer properties

Antimicrobial and chemotherapy resistance are escalating medical problem of paramount importance. Yet, research for novel antimicrobial and anticancer agents remains lagging behind. With their reported medical applications, DNA minor groove binders (MGBs) are worthy of exploration. In this study, the approach of structure-based drug design was implemented to generate 11 MGB compounds including a novel class of bioactive alkyne-linked MGBs. The NCI screening protocol was utilized to evaluate the antitumor activity of the target MGBs. Furthermore, a variety of bactericidal, cytopathogenicity, MIC90, and cytotoxicity assays were carried out using these MGBs against 6 medically relevant bacteria: Salmonella enterica, Escherichia coli, Serratia marcescens, Bacillus cereus, Streptococcus pneumoniae and Streptococcus pyogenes. Moreover, molecular docking, molecular dynamic simulations, DNA melting, and isothermal titration calorimetry (ITC) analyses were utilized to explore the binding mode and interactions between the most potent MGBs and the DNA duplex d(CGACTAGTCG)2. NCI results showed that alkyne-linked MGBs (26 & 28) displayed the most significant growth inhibition among the NCI-60 panel. In addition, compounds MGB3, MGB4, MGB28, and MGB32 showed significant bactericidal effects, inhibited B. cereus and S. enterica-mediated cytopathogenicity, and exhibited low cytotoxicity. MGB28 and MGB32 demonstrated significant inhibition of S. pyogenes, whereas MGB28 notably inhibited S. marcescens and all four minor groove binders significantly inhibited B. cereus. The ability of these compounds to bind with DNA and distort its groove dimensions provides the molecular basis for the allosteric perturbation of proteins-DNA interactions by MGBs. This study shed light on the mechanism of action of MGBs and revealed the important structural features for their antitumor and antibacterial activities, which are important to guide future development of MGB derivatives as novel antibacterial and anticancer agents.

Bimetallic interface on an optical microfiber nucleic acid sensor: Early detection in body fluids

Nucleic acid detection, which is not limited by the “window period”, is an efficacious approach for detecting human immunodeficiency virus (HIV) infection at an early time. Here, method with a short turn-around time, portability, connectivity and cost-effectiveness is developed to suppress HIV disease in the early stage, especially in remote areas. Specifically, an optical microfiber sensor enhanced by a bimetallic interface is developed for the HIV-related DNA detection in body fluids. A bimetallic interface is constructed on the optical microfiber surface to ensure that target nucleic acid hybridization occurs between two gold nanostructures where the evanescent field is strongest. The selective enhancement of the evanescent field at the binding site prevents nonspecific amplification of the whole fiber surface, which may lead to false-positive results. With the enhancement in the bimetallic interface, the sensor can detect HIV-related DNA at concentrations from 1 aM to 0.1 pM, with a limit of detection (LOD) of 0.3 aM. The portability, flexibility and selectivity of the sensor enable its application in detecting in several body fluids, such as whole serum and artificial saliva, with LODs of 1.15 aM and 1.64 aM, respectively. This work provides a powerful tool for diagnosing HIV in early stages. Ultrasensitive nucleic acid detection in artificial saliva also provides a possibility for noninvasive detection.

Designer peptide-DNA cytoskeletons regulate the function of synthetic cells.

The bottom-up engineering of artificial cells requires a reconfigurable cytoskeleton that can organize at distinct locations and dynamically modulate its structural and mechanical properties. Here, inspired by the vast array of actin-binding proteins and their ability to reversibly crosslink or bundle filaments, we have designed a library of peptide-DNA crosslinkers varying in length, valency and geometry. Peptide filaments conjoint through DNA hybridization give rise to tactoid-shaped bundles with tunable aspect ratios and mechanics. When confined in cell-sized water-in-oil droplets, the DNA crosslinker design guides the localization of cytoskeletal structures at the cortex or within the lumen of the synthetic cells. The tunable spatial arrangement regulates the passive diffusion of payloads within the droplets and complementary DNA handles allow for the reversible recruitment and release of payloads on and off the cytoskeleton. Heat-induced reconfiguration of peptide-DNA architectures triggers shape deformations of droplets, regulated by DNA melting temperatures. Altogether, the modular design of peptide-DNA architectures is a powerful strategy towards the bottom-up assembly of synthetic cells.

Replicative senescence and high glucose induce the accrual of self-derived cytosolic nucleic acids in human endothelial cells.

Recent literature shows that loss of replicative ability and acquisition of a proinflammatory secretory phenotype in senescent cells is coupled with the build-in of nucleic acids in the cytoplasm. Its implication in human age-related diseases is under scrutiny. In human endothelial cells (ECs), we assessed the accumulation of intracellular nucleic acids during in vitro replicative senescence and after exposure to high glucose concentrations, which mimic an in vivo condition of hyperglycemia. We showed that exposure to high glucose induces senescent-like features in ECs, including telomere shortening and proinflammatory cytokine release, coupled with the accrual in the cytoplasm of telomeres, double-stranded DNA and RNA (dsDNA, dsRNA), as well as RNA:DNA hybrid molecules. Senescent ECs showed an activation of the dsRNA sensors RIG-I and MDA5 and of the DNA sensor TLR9, which was not paralleled by the involvement of the canonical (cGAS) and non-canonical (IFI16) activation of the STING pathway. Under high glucose conditions, only a sustained activation of TLR9 was observed. Notably, senescent cells exhibit increased proinflammatory cytokine (IL-1β, IL-6, IL-8) production without a detectable secretion of type I interferon (IFN), a phenomenon that can be explained, at least in part, by the accumulation of methyl-adenosine containing RNAs. At variance, exposure to exogenous nucleic acids enhances both IL-6 and IFN-β1 expression in senescent cells. This study highlights the accrual of cytoplasmic nucleic acids as a marker of senescence-related endothelial dysfunction, that may play a role in dysmetabolic age-related diseases.

Deep eutectic solvent-assisted synthesis of gold nanoflowers supported on glassy carbon electrode for DNA sensor application

Gold nanoflowers (AuNF) were synthesized on a glassy carbon electrode via a one-step, eco-friendly protocol in deep eutectic solvent (DES) of choline choloride and urea, called reline, for label-free detection of DNA hybridization. DES is eco-friendly, low-cost, biocompatible, and nontoxic, and it can be used as an electrolyte to synthesize nanomaterials by using the electrochemical method. In this protocol, highly branched and stable AuNFs were obtained without using any surfactants for DNA sensor application. The electrochemical performance of the AuNF-modified electrode was studied by cyclic voltammetry and electrochemical impedance spectroscopy. Under optimal conditions, the AuNF-based DNA biosensor exhibited a sensitivity of 294.9 Ω nM−1cm−2 and 218 μA nM−1cm−2 and a limit of detection (LOD) of 10−9 M. The remarkable sensitivity and low LOD could be attributed to the good conductivity of AuNFs for accelerating electron transfer, resulting in obvious signal amplification. The DNA biosensor showed good reproducibility (RSD <3.65 %) and acceptable stability and selectivity. Its excellent performance in DNA detection suggested that the proposed electrochemical DNA sensor has great application potential in clinical diagnosis.