G-rich Sequences Research Articles

Applying Machine Learning and SERS for Precise Typing of DNA Secondary Structures.

Surface-enhanced Raman spectroscopy (SERS) has been demonstrated as an effective method for elucidating secondary structural characteristics of DNA. However, the inherent complexity of the DNA conformation and the lack of SERS samples pose challenges for identifying numerous secondary structures. To address these issues, a synergistic method integrating machine learning with SERS was proposed so as to analyze the SERS spectra of 54 well-defined conformational oligonucleotides, namely, G-quadruplex (G4), i-motif (iM), double-strand (DS), and single-strand (SS) configurations. Principal component analysis (PCA) effectively segregated the oligonucleotides into three distinct conformational groups (G4s, iMs, and others). Furthermore, linear discriminant analysis (LDA), K-nearest neighbor (KNN), and support vector machine (SVM) approaches were utilized to improve the typing accuracy of 54 trained sequences. This enabled the correct classification of the structures of five untrained sequences, as well as the identification of the predominant conformations including G4, iM, and DS formed by two complementary G-rich and C-rich sequences in acidic and neutral pH conditions. The results of this study demonstrated the potential of the proposed methodology for rapid screening and prediction of secondary DNA conformations.

Construction of a chemiluminescent biosensor based on enzymatic extension and click chemistry for sensitive measurement of MGMT activity in human breast tissues

O6-methylguanine methyltransferase (MGMT) is responsible for dealkylation of naturally occurring O6-methylguanines, and it is closely related with DNA replication, transcription, and cancers. Herein, we develop a chemiluminescent biosensor based on enzymatic extension and click chemistry for sensitive measurement of MGMT activity. When MGMT is present, the MGMT-catalyzed demethylation reaction initiates the cleavage of biotinylated dumbbell probes by PvuII restrictive enzyme, releasing two DNA fragments with 3′-OH end. The resultant DNA fragments can trigger terminal transferase (TdT)- and click chemistry-assisted isothermal amplification to obtain abundant G-rich sequences. The G-rich sequences can be captured by magnetic beads to produce a high chemiluminescence signal. This biosensor can greatly amplify the chemiluminescence signal, facilitating label-free and template-free measurement of MGMT. Especially, the introduction of dumbbell probe and PvuII enzyme can efficiently eliminate the false positive and improve the assay specificity. This biosensor possesses high sensitivity with a detection limit of 1.4 × 10−9 ng/μL, and it may accurately quantify the intracellular MGMT. Importantly, this biosensor can be used to screen the MGMT inhibitors and distinguish the MGMT level in breast tumor tissues and normal tissues, with great potential in drug discovery and cancer diagnosis.

Excellent properties of NaF and NaBr induced DNA/gold nanoparticle conjugation system: Better stability, shorter modified time, and higher loading capacity

The functionalization of gold nanoparticle (AuNP) is the key procedure for the biochemical and biomedical application. The conventional salt-aging method requires the stepwise additions of NaCl and excessive thiolated DNA, mainly due to the poor tolerance of the DNA/AuNP mixture toward NaCl. Herein, we found that NaF is capable of improving the stability for the modification of AuNP with different bases of DNA sequences (poly A/T/C/G), and allows for adding up with a high concentration of 200 mM at one time, which greatly reduces the total modification time to 0.5–1 h. Intriguingly, the introduction of NaBr effectively increases the DNA loading capacity. Besides the advantages of NaF and NaBr, the modification performance is improved via the introduction of the oligo A/T spacer for the G-rich DNA sequences. Furthermore, this method shows the superiority to another two methods (pH 3-based and salt-aging) in terms of the loading capacity or sequence components.

A Fluorescence Enhancement Sensor Based on Silver Nanoclusters Protected by Rich-G-DNA for ATP Detection.

In this study, a turn-on fluorescence sensor for the detection of adenosine 5'-triphosphate (ATP) was developed and tested using ATP-DNA2-Ag NCs. The results showed that the fluorescence of ATP-DNA2-Ag NCs was significantly enhanced with the addition of ATP. The fluorescence enhancement was a result of the specific binding activity of the ATP aptamer and ATP, which caused G-rich sequences to approach the dark DNA-Ag NCs, owing to the alteration in ATP aptamer conformation. The proposed sensor demonstrated a good linear range of 18-42 mM and a limit of detection (LOD) of 2.8 μM. The sensor's features include sensitivity, selectivity, and simple operation. In addition, the proposed sensor successfully measured ATP in 100-fold diluted fetal bovine serum.

Fluorescent and polarity-switchable photoelectrochemical dual-mode homogeneous sensing platform for ultrasensitive kanamycin detection based on EXO III-driven signal amplification.

Afluorescent and photoelectrochemical (PEC) dual-mode biosensor based on target biorecognition-triggered cyclic amplification was constructed for Kana detection. With the assistance of the catalyzed reaction of exonuclease III, a Kana-aptamer DNA duplex was designed for conducting the cyclic release of G-rich DNA sequence as well as output DNA S2. The released G-rich sequence triggers the fluorescence (FL) of thioflavin T (ThT), the intensity of which is positively correlated with the Kana concentration. The linear range is 0.2 to 30nM, and the detection limit reaches 0.07nM. Simultaneously, the released output DNA S2 was captured by Fe3O4@CdTe-probe ssDNA and then combined with methylene blue to realize the transduction of polarity-reversed PEC signal, leading to the sensitive detection of Kana with a linear range of 0.2 to 40nM and a calculated detection limit of 0.2nM. The outstanding performance endows the dual-mode biosensor a promising prospect for practical application.

Simultaneous detection of CaMV35S and NOS using fluorescence sensors with dual-emission silver nanoclusters and catalytic hairpin amplification strategy.

Adual-emission fluorescent biosensing method was developed for simultaneous determinationof CaMV35S and NOS in genetically modified(GM) plants. Two designed hairpin DNA (H1, H2) sequences were used as templates to synthesize H1-AgNCs (λex = 570nm, λem = 625nm) and H2-AgNCs (λex = 470nm, λem = 555nm). By using H1-AgNCs and H2-AgNCs as dual-signal tags, combined with signal amplification strategy of magnetic separation to reduce background signal and an enzyme-free catalytic hairpin assembly (CHA) signal amplification strategy, a novel multi-target fluorescent biosensor was fabricated to detect multiple targets based on FRET between signal tags (donors) and magnetic Fe3O4 modified graphene oxide (Fe3O4@GO, acceptors). In the presence of the target NOS and CaMV35S, the hairpin structures of H1 and H2 can be opened respectively, and the exposed sequences will hybridize with the G-rich hairpin sequences HP1 and HP2 respectively, displacing the target sequences to participate in the next round of CHA cycle. Meanwhile, H1-HP1 and H2-HP2 double-stranded DNA sequences (dsDNA) were formed, resulting in the desorption of dsDNA from the surface of Fe3O4@GO due to weak π-π interaction between dsDNA and Fe3O4@GO and leading to the fluorescence recovery of AgNCs. Under optimal conditions, the linear ranges of this fluorescence sensor were 5 ~ 300nmol L-1 for NOS and 5 ~ 200nmol L-1 CaMV35S, and the LODs were 0.14nmol L-1 and 0.18nmol L-1, respectively. In addition, the fluorescence sensor has good selectivity for the detection of NOS and CaMV35S in GM soybean samples, showing the potential applications in GM screening.

Mitochondria-Targeted Ratiometric KLA-Tweezers for Fluorescence Imaging of Potassium Ions.

Intracellular potassium ions play a crucial role in cellular homeostasis associated with physiological and pathological processes. It is still challenging but definitely crucial to precisely and dynamically image subcellular K+. Herein, the first mitochondria-targeted DNA tweezers (KLA-tweezers) were developed for the fluorescence imaging of mitochondrial K+ with high selectivity and accuracy. The proposed KLA-tweezers consisted of two DNA double-crossover (DX) motifs, a K+-aptamer, and a mitochondria-targeted peptide (KLA) which was conjugated at the rear of DX arms via complementary base pairing. Upon contact with K+, the K+-aptamer experienced a conformational change from an open-chain G-rich sequence to a G-quadruplex with a compact conformation, which was reflected by the Förster resonance energy transfer process between Cy3 and Cy5 labeled at the end of the DX arms. The open and closed states of the tweezers before and after KLA modification were confirmed by gel electrophoresis and fluorescence spectra together with atomic force microscopy (AFM) and transmission electron microscopy (TEM) images, suggesting the KLA assembly had no effect on the morphology change. The proposed tweezers and KLA-tweezers showed low cytotoxicity, excellent selectivity, and good reversibility upon alternating addition of 18-crown-6 and K+. Besides, the KLA-tweezers exhibited outstanding stability and accurate mitochondria location ability. Upon stimulation of ATP or nigericin, intracellular fluorescence imaging of mitochondrial K+ dynamics was successfully achieved. This strategy has broad prospects as a general optical sensing platform for other metal ions or organelles in living cells.

Interlocking G-Quadruplexes Using a G-Triad•G Connection: Implications for G-Wire Assembly.

G-quadruplexes are noncanonical structures of nucleic acids formed mainly by G-rich sequences and play crucial roles in important cellular processes. They are also increasingly used in nanotechnology for their valuable properties. Various unexpected structures of G-quadruplexes have been solved recently, including a stable G-quadruplex lacking one guanine in the G-tetrad core, harboring a vacant site. In this study, we demonstrate the interlocking of two intramolecular G-quadruplexes: one containing a vacant site (4n - 1) and the other with an unbound guanine (4n + 1). These G-quadruplexes interact through a G-triad-G connection with unprecedented 5'-3' stacking. Using these interconnection properties, we have identified a sequence capable of self-assembling into G-wires in K+ solutions with potential nanotechnological applications.

Capture of RNA G-quadruplex structures using an l-RNA aptamer.

G-quadruplexes (dG4 and rG4) are nucleic acid secondary structures formed by the self-assembly of certain G-rich sequences, and they have distinctive chemical properties and play crucial roles in fundamental biological processes. Small molecule G4 ligands were shown to be crucial in characterizing G4s and understanding their functions. Nevertheless, concerns regarding the specificity of these synthetic ligands for further investigation of G4s, especially for rG4 isolation purposes, have been raised. In comparison to G4 ligands, we propose a novel magnetic bead-based pulldown assay that enables the selective capture of general rG4s using functionalized l-Apt.4-1c from both simple buffer and complex media, including total RNA and the cell lysate. We found that our l-RNA aptamer can pulldown general rG4s with a higher efficiency and specificity than the G4 small molecule ligand BioTASQ v.1 in the presence of non-target competitors, including dG4 and non-G4 structures. Our findings reveal that biotinylated l-aptamers can serve as effective molecular tools for the affinity-based enrichment of rG4 of interest using this new assay, which was also verified by quantitative reverse transcription-polymerase chain reaction (RT-qPCR) on endogenous transcripts. This work provides new and important insights into rG4 isolation using a functionalized l-aptamer, which can potentially be applied in a transcript-specific or transcriptome-wide manner in the future.

Cockayne Syndrome Linked to Elevated R-Loops Induced by Stalled RNA Polymerase II during Transcription Elongation

Mutations in the Cockayne Syndrome group B (CSB) gene cause cancer in mice, but premature aging and severe neurodevelopmental defects in humans. CSB, a member of the SWI/SNF family of chromatin remodelers, plays diverse roles in regulating gene expression and transcription-coupled nucleotide excision repair (TC-NER); however, these functions do not explain the distinct phenotypic differences observed between CSB-deficient mice and humans. During investigating Cockayne Syndrome-associated genome instability, we uncover an intrinsic mechanism that involves elongating RNA polymerase II (RNAPII) undergoing transient pauses at internal T-runs where CSB is required to propel RNAPII forward. Consequently, CSB deficiency retards RNAPII elongation in these regions, and when coupled with G-rich sequences upstream, exacerbates genome instability by promoting R-loop formation. These R-loop prone motifs are notably abundant in relatively long genes related to neuronal functions in the human genome, but less prevalent in the mouse genome. These findings provide mechanistic insights into differential impacts of CSB deficiency on mice versus humans and suggest that the manifestation of the Cockayne Syndrome phenotype in humans results from the progressive evolution of mammalian genomes.

Pattern Recognition of Alkaloids by Inhibiting the Catalytic Activity of Dopzymes for Dopamine.

Exploiting the specific recognition probe for all of the biomolecules is difficult in "lock-and-key" biosensors. The cross-reaction or the semispecific probe in pattern recognition mode is an alternative strategy through extracting a multidimensional signal array from recognition elements. Here, we design a pattern recognition sensor array based on the alkaloid-inhibited catalytic activity of dopzymes for the discrimination and determination of six alkaloids. In this sensor array, three different G-rich sequences, i.e., G-triplex (G3), G-quadruplex (GQ1), and the G-quadruplex dimer (2GQ1) possessing various peroxidase activities, conjugated with a dopamine aptamer and the dopzymes (G3-d-apt, GQ1-d-apt, and 2GQ1-d-apt) were obtained with an enhanced catalytic performance for the substrate. Through the interactions between six target alkaloids and G3, GQ1, and 2GQ1 regions, the pattern signal (6 alkaloids × 3 dopzymes × 5 replicates) was obtained from the diverse inhibited effect for the dopzyme activity. In virtue of the statistical method principal component analysis (PCA), the data array was projected into a new dimensional space to acquire the three-dimensional (3D) canonical scores and grouped into their respective clusters. The sensor array exhibited an outstanding discrimination and classification capability for six alkaloids with different concentrations with 100% accuracy. In addition, the nonspecific recognition elements of the sensor array showed high selectivity even though other alkaloids with similar structures to targets existed in the samples. Importantly, the levels of the six targets can be analyzed by the most influential discrimination factor, which represented the vector with the highest variance, evidencing that the sensor array has potential in drug screening and clinical treatment.

Functional Analysis of GRSF1 in the Nuclear Export and Translation of Influenza A Virus mRNAs.

Influenza A viruses (IAV) utilize host proteins throughout their life cycle to infect and replicate in their hosts. We previously showed that host adaptive mutations in avian IAV PA help recruit host protein G-Rich RNA Sequence Binding Factor 1 (GRSF1) to the nucleoprotein (NP) 5' untranslated region (UTR), leading to the enhanced nuclear export and translation of NP mRNA. In this study, we evaluated the impact of GRSF1 in the viral life cycle. We rescued and characterized a 2009 pH1N1 virus with a mutated GRSF1 binding site in the 5' UTR of NP mRNA. Mutant viral growth was attenuated relative to pH1N1 wild-type (WT) in mammalian cells. We observed a specific reduction in the NP protein production and cytosolic accumulation of NP mRNAs, indicating a critical role of GRSF1 in the nuclear export of IAV NP mRNAs. Further, in vitro-transcribed mutated NP mRNA was translated less efficiently than WT NP mRNA in transfected cells. Together, these findings show that GRSF1 binding is important for both mRNA nuclear export and translation and affects overall IAV growth. Enhanced association of GRSF1 to NP mRNA by PA mutations leads to rapid virus growth, which could be a key process of mammalian host adaptation of IAV.

Target Recognition Triggered Split DNAzyme based Colorimetric Assay for Direct and Sensitive Methicillin-Resistance Analysis of Staphylococcus aureus.

The accurate and rapid detection of methicillin-resistant Staphylococcus aureus (MRSA) holds significant clinical importance. This work presents a new method for detecting methicillin-resistant Staphylococcus aureus (S. aureus) in clinical samples. The method uses an aptamer-based colorimetric assay that combines a recognizing probe to identify the target and split DNAzyme to amplify the signal, resulting in a highly sensitive and direct analysis of methicillin-resistance. The identification of the PBP2a protein on the membrane of S. aureus in clinical samples leads to the allosterism of the recognizing probe, and thus provides a template for the proximity ligation of split DNAzyme. The proximity ligation of split DNAzyme forms an intact DNAzyme to identify the loop section in the L probe and generates a nicking site to release the loop sequence ("3" and "4" fragments). The "3" and "4" fragments forms an intact sequence to induce the catalytic hairpin assembly, exposing the G-rich section. The released the G-rich sequence of LR probe induces the formation of G-quadruplex-hemin DNAzyme as a colorimetric signal readout. The absorption intensity demonstrated a strong linear association with the logarithm of the S. aureus concentration across a wide range of 5 orders of magnitude dynamic range under the optimized experimental parameters. The limit of detection was calculated to be 23 CFU/ml and the method showed high selectivity for MRSA.

Redox-responsive degradation of DNA@MnO2 nanoprobe triggering DNAzyme recycling amplification for sensitive and label-free detection of glutathione

In this work, a novel fluorescence nanoprobe based on MnO2 nanosheets and DNAzyme cyclic amplification was developed for label-free detection of GSH. In our design, the DNA@MnO2 nanoprobe was facilely prepared through the adsorption of DNA probes on the surface of MnO2 nanosheets. Importantly, MnO2 nanosheets in the DNA@MnO2 nanoprobe can act as the recognizer of analytical target GSH. In the presence of GSH, the DNA@MnO2 nanoprobe are decomposed owing to redox-responsive reaction between GSH and MnO2 nanosheets, resulting in the generation of abundant Mn2+ and the desorption of DNA probes. Subsequently, the Mn2+-dependent DNAzyme recycling amplification was rapidly initiated and a large number of the blocked G-rich sequences in DNA probes was released. The G-rich sequences were efficiently folded into G-quadruplex structures, which can remarkably light up the fluorescence intensity of thioflavin T (ThT). Experimental data demonstrated that the DNA@MnO2 nanoprobes for GSH detection have a wide linear range from 0 to 2000 µM. Moreover, this fluorescence sensing strategy was successfully applied for the detection of GSH in complex biological media with satisfying results, providing a simple, facile and cost-efficient nanoplatform for GSH-related clinical disease diagnosis.

Identification of a conserved G-quadruplex within the E165R of African swine fever virus (ASFV) as a potential antiviral target

African swine fever virus (ASFV) is a double-stranded DNA arbovirus with high transmissibility and mortality rates. It has caused immense economic losses to the global pig industry. Currently, no effective vaccines or medications are to combat ASFV infection. G-quadruplex (G4) structures have attracted increasing interest because of their regulatory role in vital biological processes. In this study, we identified a conserved G-rich sequence within the E165R gene of ASFV. Subsequently, using various methods, we verified that this sequence could fold into a parallel G-quadruplex (G4). In addition, the G4-stabilizers pyridostatin (PDS) and 5,10,15,20-tetrakis-(N-methyl-4-pyridyl) porphin (TMPyP4) can bind and stabilize this G4 structure, thereby inhibiting E165R gene expression, and the inhibitory effect is associated with G4 formation. Moreover, the G4 ligand PDS substantially impeded ASFV proliferation in Vero cells by reducing gene copy number and viral protein expression. These compelling findings suggest that G4 structures may represent a promising and novel antiviral target against ASFV.

A Fluorescence Light-Up 3D DNA Walker Driven and Accelerated by Endogenous Adenosine-5'-triphosphate for Sensitive and Rapid Label-Free MicroRNA Detection and Imaging in Living Cells.

Herein, a fluorescence light-up 3D DNA walker (FLDW) was powered and accelerated by endogenous adenosine-5'-triphosphate (ATP) molecules to construct a biosensor for sensitive and rapid label-free detection and imaging of microRNA-221 (miRNA-221) in malignant tumor cells. Impressively, ATP as the driving force and accelerator for FLDW could significantly accelerate the operation rate of FLDW, reduce the likelihood of errors in signaling, and improve the sensitivity of detection and imaging. When FLDW was initiated by output DNA H1-op transformed by target miRNA-221, G-rich sequences in the S strand, anchored to AuNP, were exposed to form G-quadruplexes (G4s), and thioflavin T (ThT) embedded in the G4s emitted intense fluorescence to realize sensitive and rapid detection of target miRNA-221. Meanwhile, the specific binding of ThT to G4 with a weak background fluorescence response was utilized to enhance the signal-to-noise ratio of the label-free assay straightforwardly and cost-effectively. The proposed FLDW system could realize sensitive detection of the target miRNA-221 in the range of 1 pM to 10 nM with a detection limit of 0.19 pM by employing catalytic hairpin assembly (CHA) to improve the conversion of the target. Furthermore, by harnessing the abundant ATP present in the tumor microenvironment, FLDW achieved rapid and accurate imaging of miRNA-221 in cancer cells. This strategy provides an innovative and high-speed label-free approach for the detection and imaging of biomarkers in cancer cells and is expected to be a powerful tool for bioanalysis, diagnosis, and prognosis of human diseases.

Nanoscale Interaction of Endonuclease APE1 with DNA.

Apurinic/apyrimidinic endonuclease 1 (APE1) is involved in DNA repair and transcriptional regulation mechanisms. This multifunctional activity of APE1 should be supported by specific structural properties of APE1 that have not yet been elucidated. Herein, we applied atomic force microscopy (AFM) to characterize the interactions of APE1 with DNA containing two well-separated G-rich segments. Complexes of APE1 with DNA containing G-rich segments were visualized, and analysis of the complexes revealed the affinity of APE1 to G-rich DNA sequences, and their yield was as high as 53%. Furthermore, APE1 is capable of binding two DNA segments leading to the formation of loops in the DNA-APE1 complexes. The analysis of looped APE1-DNA complexes revealed that APE1 can bridge G-rich segments of DNA. The yield of loops bridging two G-rich DNA segments was 41%. Analysis of protein size in various complexes was performed, and these data showed that loops are formed by APE1 monomer, suggesting that APE1 has two DNA binding sites. The data led us to a model for the interaction of APE1 with DNA and the search for the specific sites. The implication of these new APE1 properties in organizing DNA, by bringing two distant sites together, for facilitating the scanning for damage and coordinating repair and transcription is discussed.

New G-Triplex DNA Dramatically Activates the Fluorescence of Thioflavin T and Acts as a Turn-On Fluorescent Sensor for Uracil-DNA Glycosylase Activity Detection.

G-triplexes are G-rich oligonucleotides composed of three G-tracts and have absorbed much attention due to their potential biological functions and attractive performance in biosensing. Through the optimization of loop compositions, DNA lengths, and 5'-flanking bases of G-rich sequences, a new stable G-triplex sequence with 14 bases (G3-F15) was discovered to dramatically activate the fluorescence of Thioflavin T (ThT), a water-soluble fluorogenic dye. The fluorescence enhancement of ThT after binding with G3-F15 reached 3200 times, which was the strongest one by far among all of the G-rich sequences. The conformations of G3-F15 and G3-F15/ThT were studied by circular dichroism. The thermal stability measurements indicated that G3-F15 was a highly stable G-triplex structure. The conformations of G3-F15 and G3-F15/ThT in the presence of different metal cations were studied thoroughly by fluorescent spectroscopy, circular dichroism, and nuclear magnetic resonance. Furthermore, using the G3-F15/ThT complex as a fluorescent probe, a robust and simple turn-on fluorescent sensor for uracil-DNA glycosylase activity was developed. This study proposes a new systematic strategy to explore new functional G-rich sequences and their ligands, which will promote their applications in diagnosis, therapy, and biosensing.

Low-background fluorescent biosensor based on graphene oxide and aptamer biorecognition for sensitive detection of kanamycin

The detection of kanamycin has attracted extensive attention in the field of food safety. A label-free, low-background fluorescent biosensor was successfully constructed based on graphene oxide and aptamer biorecognition in this paper for kanamycin detection in milk samples. In this biosensor, the integrated probe with the aptamer sequence of kanamycin and the G-rich sequence was designed. The aptamer sequence of kanamycin was used as the recognition part, the G-quadruplex/N-methyl mesoporphyrin IX (NMM) complex as the fluorescent probe, and graphene oxide as the fluorescent quencher. Initially, the integrated probe could be adsorbed on the graphene oxide surface through π-π stacking interactions, while the fluorescence of NMM could be efficiently quenched because of the resonance energy transfer between NMM and graphene oxide, which was beneficial to the low background signal of the system. During the detection process, the aptamer sequence preferred to bind to kanamycin, forming aptamer-kanamycin complex. At the same time, the G-rich sequence folded into G-quadruplex structure that specifically bound to NMM under the action of K+, forming G-quadruplex/NMM complex. Under the synergistic effects of the two complexes, the integrated probe with aptamer-kanamycin complex and G-quadruplex/NMM complex was released from the graphene oxide surface due to the steric hindrance effect, restoring the strong fluorescence signal. By combining graphene oxide and G-quadruplex/NMM complex, the fluorescent biosensor could effectively achieve label-free and low-background detection of kanamycin. The linear calibration curve was in the range of 1–400 nM with a detection limit of 0.60 nM for kanamycin. It was successfully applied to analyze raw milk samples (spiked kanamycin) with recoveries of 98.33–101.25 % and also exhibited high selectivity for kanamycin. The experimental data showed that the biosensor was a potentially powerful tool for monitoring food safety.

A G-quadruplex/thioflavin T-based label-free biosensor to detect ClO− in stress-induced hypertension

Hypochlorous acid (HClO), as an essential reactive oxygen species (ROS) in biological systems, plays a pivotal role in processes of physiology and pathology. Abnormal fluctuations in HClO concentration can lead to various diseases, such as inflammation, cardiovascular diseases, and neurodegeneration. Therefore, developing an approach to rapidly and sensitively quantify ClO− content is vital to biomedicine development and bioassays. Herein, we fabricated a novel “turn-on” label-free fluorescence DNA probe to specifically detect hypochlorite ion (ClO−) based on G-quadruplex formation. To this end, we designed a G-rich signal DNA sequence (S-DNA) and a block DNA sequence (B-DNA), followed by the introduction of ClO−-responsive phosphorothioate (PS) into B-DNA. In the absence of ClO−, B-DNA hybridized with S-DNA, preventing G-quadruplex formation from S-DNA; this resulted in the relatively low fluorescence intensity of ThT. Once ClO− was added, the hydrolysis between PS and ClO− split the B-DNA into two fragments, resulting in B-DNA breaking away from S-DNA, allowing G-quadruplex formation from S-DNA and increasing the fluorescence intensity of ThT. Using this method, we can detect ClO− without the interference of additional reactive oxygen species. The detection limit of ClO− was as low as 10 nM. Furthermore, this method facilitates the detection of ClO− within the tissues of rats with stress-induced hypertension.