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Cyclic GMP-AMP synthase (cGAS) is a major cytosolic DNA sensor that plays a significant role in innate immunity. Upon binding to double stranded DNA (dsDNA), cGAS utilizes GTP and ATP to synthesize the second messenger cyclic GMP-AMP (cGAMP). The cGAMP then binds to the adapter protein stimulator of interferon genes (STING) in the endoplasmic reticulum, resulting in the activation of the transcription factor interferon regulatory factor 3 (IRF3) and subsequent induction of type I interferon. An important question is how cGAS distinguishes between self and non-self DNA. While cGAS binds to the phosphate backbone of DNA without discrimination, its activation is influenced by physical features such as DNA length, inter-DNA distance, and mechanical flexibility. This suggests that the recognition of DNA by cGAS may depend on these physical features. In this article we summarize the recent progress in research on cGAS-STING pathway involved in antiviral defense, cellular senescence and anti-tumor response, and focus on DNA recognition mechanisms based on the physical features.

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Dendritic cells (DCs) are involved in the initiation and maintenance of immune responses against malignant cells by recognizing conserved pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) through pattern recognition receptors (PRRs). According to recent studies, tumor cell-derived DNA molecules act as DAMPs and are recognized by DNA sensors in DCs. Once identified by sensors in DCs, these DNA molecules trigger multiple signaling cascades to promote various cytokines secretion, including type I IFN, and then to induce DCs mediated antitumor immunity. As one of the potential attractive strategies for cancer therapy, various agonists targeting DNA sensors are extensively explored including the combination with other cancer immunotherapies or the direct usage as major components of cancer vaccines. Moreover, this review highlights different mechanisms through which tumor-derived DNA initiates DCs activation and the mechanisms through which the tumor microenvironment regulates DNA sensing of DCs to promote tumor immune escape. The contributions of chemotherapy, radiotherapy, and checkpoint inhibitors in tumor therapy to the DNA sensing of DCs are also discussed. Finally, recent clinical progress in tumor therapy utilizing agonist-targeted DNA sensors is summarized. Indeed, understanding more about DNA sensing in DCs will help to understand more about tumor immunotherapy and improve the efficacy of DC-targeted treatment in cancer.

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Cervical cancer is caused by changes in the cervix that lead to precancerous cells and eventually progress to cancer. Human papillomavirus (HPV) infections are the primary cause of cervical cancer. Early detection of HPV is crucial in preventing cervical cancer, and regular screening for HPV infection can identify cell changes before they develop into cancer. While Pap smear tests are reliable for cervical cancer screening, they are critical, expensive, and labor-intensive. Therefore, researchers are focusing on identifying blood-based biomarkers using biosensors for cervical cancer screening. HPV strains 16, 45, and 18 are common culprits in cervical cancer. This study aimed to develop an HPV-16 DNA biosensor on a zeolite-iron oxide (zeolite-IO) modified interdigitated electrode (IDE) sensor. The DNA probe was immobilized on the IDE through amine-modified zeolite-IO, enhancing the hybridization of the target and DNA probe. The detection limit of the DNA-DNA duplex was found to be 7.5 pM with an R2 value of 0.9868. Additionally, control experiments with single and triple mismatched sequences showed no increase in current responses, and the identification of target DNA in a serum-spiked sample indicated specific and selective target identification.

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BACKGROUND: High-efficiency and highly reliable analysis of microRNAs (miRNAs) in bodily fluids highlights its significance to be extensively utilized as candidates for non-invasive "liquid biopsy" approaches. DNA biosensors based on strand displacement amplification (SDA) methods have been successfully designed to detect miRNAs given the efficiently amplified and recycled of the target sequences. However, the unpredictable DNA framework and heavy reliance on free diffusion or random reactant collisions in existing approaches lead to delayed reaction kinetics and inadequate amplification. Thus, it is crucial to create a modular probe with a controlled structure, high local concentration, and ease of synthesis. RESULTS: Inspired by the natural spatial-confinement effect based on a well-known streptavidin-biotin interaction, we constructed a protein-DNA hybrid, named protein-scaffolded DNA tetrads (PDT), which consists of four biotinylated Y-shaped DNA (Y-DNA) surrounding a streptavidin protein center via a streptavidin-biotin bridge. The streptavidin-biotin recognition system significantly increased the local concentration and intermolecular distance of the probes to achieve enhanced reaction efficiency and kinetics. The PDT-based assay starts with the target miRNA binding to Y-DNA, which disassembles the Y-DNA structures into three types of hairpin-shaped structures via self-primed strand displacement amplification (SPSDA) and generates remarkable fluorescence signal that is proportional to the miRNA concentration. Results demonstrated that PDT enabled a more efficient detection of miRNA-21 with a sensitivity of 1 fM. Moreover, it was proven reliable for the detection of clinical serum samples, suggesting great potential for advancing the development of rapid and robust signal amplification technologies for early diagnosis. SIGNIFICANCE: This simple yet robust system contributes to the early diagnosis of miR-21 with satisfactory sensitivity and specificity, and display a significantly improved nuclease resistance owing to their unique structure. The results suggested that the strategy is expected to provide a promising potential platform for tumor diagnosis, prognosis and therapy.

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Cytosolic DNA sensors (CDSs) recognize DNA molecules that are abnormally located in the cytosol, thus leading to the activation of the stimulator of interferon genes (STING) and the induction of type 1 interferon. In turn, type 1 interferon evokes defensive reactions against viral infections and activates the immune system; therefore, the use of agonists of CDSs as cancer therapeutics and vaccine adjuvants is expected. Double-stranded DNA molecules with dozens to thousands of bases derived from bacteria and viruses are agonists of CDSs. However, DNA is a water-soluble molecule with a high molecular weight, resulting in poor cellular uptake and endosomal escape. In contrast, long single-stranded DNA (lssDNA) obtained by rolling circle amplification is efficiently taken up and localized to endosomes. Here we constructed a CDS-targeting lssDNA via the facilitation of its intracellular transport from endosomes to the cytosol. An endosome-disrupting GALA peptide was used to deliver the lssDNA to the cytosol. A peptide-oligonucleotide conjugate (POC) was successfully obtained via the conjugation of the GALA peptide with an oligonucleotide complementary to the lssDNA. By hybridization of the POC to the complementary lssDNA (POC/lssDNA), the CDS-STING pathway in dendritic cells was efficiently stimulated. GALA peptide-conjugated DNA seems to be a helpful tool for the delivery of DNA to the cytosol.

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The presence of acylamide (AA) in large group of food products and its health hazards have been confirmed by scientists. In this study, a simple and innovative biosensor for AA determination was designed based on single-stranded DNA (ssDNA) with partial guanine and GelRed. The idea of this biosensor is based on the formation of AA-ssDNA adduct through the strong binding interaction between AA and guanine base of ssDNA, which subsequently inhibits the interaction of ssDNA and GelRed, leading to a weak fluorescence intensity. The binding interaction between AA and ssDNA was confirmed by UV-Vis absorption spectrometry and fluorescence intensity. Under optimum conditions, the designed biosensor exhibited excellent linear response in range of 0.01-95 mM, moreover it showed high selectivity toward AA. The limit of detection was 0.003 mM. This biosensor was successfully applied for the determination of AA in water extract of potato fries and coffee in the range of 0.05-100 mM with LOD of 0.01 mM and 0.05-95 mM with LOD of 0.004 mM, respectively.

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A novel electrochemical DNA sensor was developed for the detection of the anthracycline drug, valrubicin, on the base of poly(Azure C) electropolymerized from the deep eutectic solvent reline and covered with adsorbed DNA from calf thymus. Biosensor assembling was performed by multiple scanning of the potential in one drop (100 µL) of the dye dissolved in reline and placed on the surface of a screen-printed carbon electrode. Stabilization of the coating was achieved by its polarization in the phosphate buffer. The electrochemical characteristics of the electron transfer were determined and compared with a similar coating obtained from phosphate buffer. The use of deep eutectic solvent made it possible to increase the monomer concentration and avoid using organic solvents on the stage of electrode modification. After the contact of the DNA sensor with valrubicin, two signals related to the intrinsic redox activity of the coating and the drug redox conversion were found on voltammogram. Their synchronous changes with the analyte concentration increased the reliability of the detection. In the square-wave mode, the DNA sensor made it possible to determine from 3 µM to 1 mM (limit of detection, 1 µM) in optimal conditions. The DNA sensor was successfully tested in the voltammetric determination of valrubicin in spiked artificial urine, Ringer-Locke solution mimicking plasma electrolytes and biological samples (urine and saliva) with a recovery of 90-110%. After further testing on clinical samples, it can find application in the pharmacokinetics studies and screening of new drugs' interaction with DNA.

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An electrochemically active polymer, polythionine (PTN), was synthesized in natural deep eutectic solvent (NADES) via multiple potential scans and characterized using cyclic voltammetry and electrochemical impedance spectroscopy (EIS). NADES consists of citric acid monohydrate, glucose, and water mixed in the molar ratio of 1:1:6. Electrodeposited PTN film was then applied for the electrostatic accumulation of DNA from salmon sperm and used for the sensitive detection of the anticancer drug epirubicin. Its reaction with DNA resulted in regular changes in the EIS parameters that made it possible to determine 1.0-100 µM of epirubicin with the limit of detection (LOD) of 0.3 µM. The DNA sensor developed was successfully applied for the detection of epirubicin in spiked samples of artificial and natural urine and saliva, with recovery ranging from 90 to 109%. The protocol of the DNA sensor assembling utilized only one drop of reactants and was performed with a minimal number of steps. Together with a simple measurement protocol requiring 100 µL of the sample, this offers good opportunities for the further use of the DNA sensor in monitoring the drug level in biological samples, which is necessary in oncology treatment and for the pharmacokinetics studies of new antitumor drugs.

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A triplex DNA electrochemical sensor based on reduced graphene oxide (rGO) and electrodeposited gold nanoparticles (EAu) was simply fabricated for Pb2+ detection. The glass carbon electrode (GCE) sequentially electrodeposited with rGO and EAu was further modified with a triplex DNA helix that consisted of a guanine (G)-rich circle and a stem of triplex helix based on T-A•T base triplets. With the existence of Pb2+, the DNA configuration which was formed via the Watson-Crick and Hoogsteen base pairings was split and transformed into a G-quadruplex. An adequate electrochemical response signal was provided by the signal indicator methylene blue (MB). The proposed sensor demonstrated a linear relationship between the differential pulse voltammetry (DPV) peak currents and the logarithm of Pb2+ concentrations from 0.01 to 100.00 µM with a detection limit of 0.36 nM. The proposed sensor was also tested with tap water, river and medical wastewater samples with qualified recovery and accuracy and represented a promising method for Pb2+ detection.

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A novel voltammetric sensor based on a self-assembled composite formed by native DNA and electropolymerized N-phenyl-3-(phenylimino)-3H-phenothiazin-7-amine has been developed and applied for sensitive determination of doxorubicin, an anthracycline drug applied for cancer therapy. For this purpose, a monomeric phenothiazine derivative has been deposited on the glassy carbon electrode from the 0.4 M H2SO4-acetone mixture (1:1 v/v) by multiple potential cycling. The DNA aliquot was either on the electrode modified with electropolymerized film or added to the reaction medium prior to electropolymerization. The DNA entrapment and its influence on the redox behavior of the underlying layer were studied by scanning electron microscopy and electrochemical impedance spectroscopy. The DNA-doxorubicin interactions affected the charge distribution in the surface layer and, hence, altered the redox equilibrium of the polyphenothiazine coating. The voltametric signal was successfully applied for the determination of doxorubicin in the concentration range from 10 pM to 0.2 mM (limit of detection 5 pM). The DNA sensor was tested on spiked artificial plasma samples and two commercial medications (recovery of 90-95%). After further testing on real clinical samples, the electrochemical DNA sensor developed can find application in monitoring drug release and screening new antitumor drugs able to intercalate DNA.

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[This corrects the article DOI: 10.3389/fmicb.2019.02627.].

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Antiviral immune responses are mainly triggered through the recognition of virus-derived nucleic acids by host-specific pattern recognition receptors (PRRs). Here, we identified and characterized homologs of human PRRs for virus-derived DNA in Bombyx mori upon infection with a nucleopolyhedrovirus (NPV), a member of the family Baculoviridae. We found that progeny virus production of B. mori NPV was promoted in B. mori cells silenced with B. mori homolog of DEAD/H box polypeptide 9 gene (Bm-DHX9), but not in cells silenced with the other examined genes. Silencing of Bm-DHX9 expression has no effect on apoptosis induction, one of the major antiviral responses in B. mori cells. We also showed that Bm-DHX9 has the ability to bind DNA containing unmethylated C-phosphate-G-motif, which are characteristic of microbial pathogens and contained in the NPV genome with high frequency. Our findings suggest that Bm-DHX9 has the potential for sensing NPV-derived DNA to induce antiviral immune responses.

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Genetic immunization is an attractive approach for prophylactic and therapeutic vaccination using synthetic vectors to deliver antigen-encoding nucleic acids. Recently, DNA delivered by a physical means or RNA by liposomes consisting of four different lipids demonstrated good protection in human phase III clinical trials and received Drugs Controller General of India and US FDA approval to protect against COVID-19, respectively. However, the development of a system allowing for efficient and simple delivery of nucleic acids while improving immune response priming has the potential to unleash the full therapeutic potential of genetic immunization. DNA-based gene therapies and vaccines have the potential for rapid development, as exemplified by the recent approval of Collategene, a gene therapy to treat human critical limb ischemia, and ZyCoV, a DNA vaccine delivered by spring-powered jet injector to protect against SARS-CoV2 infection. Recently, we reported amphiphilic block copolymer 704 as a promising synthetic vector for DNA vaccination in various models of human diseases. This vector allows dose sparing of antigen-encoding plasmid DNA. Here, we report the capacity of 704-mediated HIV and anti-hepatocellular carcinoma DNA vaccines to induce the production of specific antibodies against gp120 HIV envelope proteins in mice and against alpha-fetoprotein antigen in non-human primates, respectively. An investigation of the underlying mechanisms showed that 704-mediated vaccination did trigger a strong immune response by (1) allowing a direct DNA delivery into the cytosol, (2) promoting an intracytoplasmic DNA sensing leading to both interferon and NF-κB cascade stimulation, and (3) inducing antigen expression by muscle cells and presentation by antigen-presenting cells, leading to the induction of a robust adaptive response. Overall, our findings suggest that the 704-mediated DNA vaccination platform is an attractive method to develop both prophylactic and therapeutic vaccines.

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Electrochemical DNA sensors can be operated in either static or flow-based detection schemes. In static schemes, manual washing steps are still necessary, resulting in a tedious and time-consuming process. In contrast, in flow-based electrochemical sensors, the current response is collected when the solution flows through the electrode continuously. However, the drawback of such a flow system is the low sensitivity due to the limited time for the interaction between the capturing element and the target. Herein, we propose a novel electrochemical capillary-driven microfluidic DNA sensor to combine the advantages of static and flow-based electrochemical detection systems into a single device by incorporating burst valve technology. The microfluidic device with a two-electrode configuration was applied for the simultaneous detection of two different DNA markers, human immunodeficiency virus-1 (HIV-1) and hepatitis C virus (HCV) cDNA, via the specific interaction between pyrrolidinyl peptide nucleic acids (PNA) probes and the DNA target. The integrated system, while requiring a small sample volume (7 µL for each sample loading port) and less analysis time, achieved good performance in terms of the limits of detection (LOD) (3SDblank/slope) and quantification (LOQ) (10SDblank/slope) at 1.45 nM and 4.79 nM for HIV and 1.20 nM and 3.96 nM for HCV, respectively. The simultaneous detection of HIV-1 and HCV cDNA prepared from human blood samples showed results that are in complete agreement with the RT‒PCR assay. The results qualify this platform as a promising alternative for the analysis of either HIV-1/HCV or coinfection that can be easily adapted for other clinically important nucleic acid-based markers.

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Electrochemical DNA sensors are highly demanded for fast and reliable determination of antitumor drugs and chemotherapy monitoring. In this work, an impedimetric DNA sensor has been developed on the base of a phenylamino derivative of phenothiazine (PhTz). A glassy carbon electrode was covered with electrodeposited product of PhTz oxidation obtained through multiple scans of the potential. The addition of thiacalix[4]arene derivatives bearing four terminal carboxylic groups in the substituents of the lower rim improved the conditions of electropolymerization and affected the performance of the electrochemical sensor depending on the configuration of the macrocyclic core and molar ratio with PhTz molecules in the reaction medium. Following that, the deposition of DNA by physical adsorption was confirmed by atomic force microscopy and electrochemical impedance spectroscopy. The redox properties of the surface layer obtained changed the electron transfer resistance in the presence of doxorubicin due to its intercalating DNA helix and influencing charge distribution on the electrode interface. This made it possible to determine 3 pM-1 nM doxorubicin in 20 min incubation (limit of detection 1.0 pM). The DNA sensor developed was tested on a bovine serum protein solution, Ringer-Locke's solution mimicking plasma electrolytes and commercial medication (doxorubicin-LANS) and showed a satisfactory recovery rate of 90-105%. The sensor could find applications in pharmacy and medical diagnostics for the assessment of drugs able to specifically bind to DNA.

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An advanced multi-parameter optical fiber sensing technology for EGFR gene detection based on DNA hybridization technology is demonstrated in this paper. For traditional DNA hybridization detection methods, temperature and pH compensation can not be realized or need multiple sensor probes. However, the multi-parameter detection technology we proposed can simultaneously detect complementary DNA, temperature and pH based on a single optical fiber probe. In this scheme, three optical signals including dual surface plasmon resonance signal (SPR) and Mach-Zehnder interference signal (MZI) are excited by binding the probe DNA sequence and pH-sensitive material with the optical fiber sensor. The paper proposes the first research to achieve simultaneous excitation of dual SPR signal and Mach-Zehnder interference signal in a single fiber and used for three-parameter detection. Three optical signals have different sensitivities to the three variables. From a mathematical point of view, the unique solutions of exon-20 concentration, temperature and pH can be obtained by analyzing the three optical signals. The experimental results show that the exon-20 sensitivity of the sensor can reach 0.07 nm nM-1, and the limit of detection is 3.27 nM. The designed sensor gives a fast response, high sensitivity, and low detection limit, which is important for the field of DNA hybridization research and for solving the problems of biosensor susceptibility to temperature and pH.

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cGAS and AIM2 are CDSs that are activated in the presence of cytosolic dsDNA and are expressed in various cell types, including immune and tumor cells. The recognition of tumor-derived dsDNA by CDSs in the cytosol of tumor-infiltrating dendritic cells (TIDCs) activates the innate and acquired immunity, thereby enhancing anti-tumor immune responses. STING is the downstream signaling effector of cGAS that induces type I interferon (IFN) signaling. Owing to their ability to activate TIDCs, STING agonists have been intratumorally injected in several clinical trials to enhance the anti-tumor immune response elicited by immune checkpoint antibodies. However, they have shown minimal effect, suggesting the importance of optimizing the dose and route of administration for STING agonists and deciphering other immune pathways that contribute to anti-tumor immune responses. Recent studies have revealed that AIM2 activity induces pro-tumor growth through multiple parallel pathways, including inhibition of STING-type I IFN signaling. Thus, AIM2 could be a potential molecular target for cancer immunotherapies. This review summarizes the current research on the roles of cGAS, STING, and AIM2 in immune cells and tumor cells in the tumor microenvironment and discusses the future prospects of anti-tumor treatment approaches based on these molecules.

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Considering the high cost and tedious process of gene sequencing, there is an urgent need to develop portable and efficient sensors for the TP53 gene. Here, we developed a novel electrochemical sensor that detected the TP53 gene using magnetic peptide nucleic acid (PNA)-modified Fe3O4/α-Fe2O3@Au nanocomposites. Cyclic voltammetry and electrochemical impedance spectroscopy confirmed the successful stepwise construction of the sensor, especially the high-affinity binding of PNA to DNA strands, which induced different electron transfer rates and resulted in current changes. Variations in the differential pulse voltammetry current observed during hybridization at different surface PNA probe densities, hybridization times, and hybridization temperatures were explored. The biosensing strategy obtained a limit of detection of 0.26 pM, a limit of quantification of 0.85 pM, and a wide linear range (1 pM-1 µM), confirming that the Fe3O4/α-Fe2O3@Au nanocomposites and the strategy based on magnetic separation and magnetically induced self-assembly improved the binding efficiency of nucleic acid molecules. The biosensor was a label-free and enzyme-free device with excellent reproducibility and stability that could identify single-base mismatched DNA without additional DNA amplification procedures, and the serum spiked experiments revealed the feasibility of the detection approach.

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Silver ion (Ag+) is one of the most common heavy metal ions that cause environmental pollution and affect human health, and therefore, its detection is of great importance in the field of analytical chemistry. Here, we report an 8-nucleotide (nt) minidumbbell DNA-based sensor (M-DNA) for Ag+ detection. The minidumbbell contained a unique reverse wobble C·C mispair in the minor groove, which served as the binding site for Ag+. The M-DNA sensor could achieve a detection limit of 2.1 nM and sense Ag+ in real environmental samples with high accuracy. More importantly, the M-DNA sensor exhibited advantages of fast kinetics and easy operation owing to the usage of an ultrashort oligonucleotide. The minidumbbell represents a new and minimal non-B DNA structural motif for Ag+ sensing, allowing for the further development of on-site environmental Ag+ detection devices.

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A novel technique based on inverted Lamb wave MEMS resonator has been exploited for the realization of a DNA biosensor. Zinc oxide based Lamb wave MEMS resonator in the inverted configuration of ZnO/SiO2/Si/ZnO is fabricated for label free and efficient detection of Neisseria meningitidis, responsible for bacterial meningitis. Meningitis remains a devastating endemic in sub-Saharan Africa. Its early detection can prevent the spread and its lethal complications. The developed biosensor shows a very high sensitivity of 310 Hz(ngµl-1)-1 and very low detection limit of 82 pgµl-1 for symmetric mode of the Lamb wave device while the antisymmetric mode shows a sensitivity of 202 Hz(ngµl-1)-1 and the limit of detection of 84 pgµl-1. This very high sensitivity and very low detection limit of the Lamb wave resonator can be attributed to very high mass loading effect on the membranous structure of Lamb wave device, unlike the bulk substrate based devices. The indigenously developed MEMS based inverted Lamb wave biosensor shows high selectivity, long shelf life and good reproducibility. The ease of operation, low processing time and possibility of wireless integration of the of the Lamb wave DNA sensor paves a path towards the promising application in the field of meningitidis detection. The use of fabricated biosensor can be extended to other viral and bacterial detection applications as well.

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