G-rich Sequences Research Articles

Stabilization of the Quadruplex-Forming G-Rich Sequences in the Rhinovirus Genome Inhibits Uncoating-Role of Na+ and K.

Rhinoviruses (RVs) are the major cause of common cold, a respiratory disease that generally takes a mild course. However, occasionally, RV infection can lead to serious complications in patients debilitated by other ailments, e.g., asthma. Colds are a huge socioeconomic burden as neither vaccines nor other treatments are available. The many existing drug candidates either stabilize the capsid or inhibit the viral RNA polymerase, the viral proteinases, or the functions of other non-structural viral proteins; however, none has been approved by the FDA. Focusing on the genomic RNA as a possible target for antivirals, we asked whether stabilizing RNA secondary structures might inhibit the viral replication cycle. These secondary structures include G-quadruplexes (GQs), which are guanine-rich sequence stretches forming planar guanine tetrads via Hoogsteen base pairing with two or more of them stacking on top of each other; a number of small molecular drug candidates increase the energy required for their unfolding. The propensity of G-quadruplex formation can be predicted with bioinformatics tools and is expressed as a GQ score. Synthetic RNA oligonucleotides derived from the RV-A2 genome with sequences corresponding to the highest and lowest GQ scores indeed exhibited characteristics of GQs. In vivo, the GQ-stabilizing compounds, pyridostatin and PhenDC3, interfered with viral uncoating in Na+ but not in K+-containing phosphate buffers. The thermostability studies and ultrastructural imaging of protein-free viral RNA cores suggest that Na+ keeps the encapsulated genome more open, allowing PDS and PhenDC3 to diffuse into the quasi-crystalline RNA and promote the formation and/or stabilization of GQs; the resulting conformational changes impair RNA unraveling and release from the virion. Preliminary reports have been published.

DSN enzyme recognition initiated rolling circle amplification (DiRCA) for accurate miRNA detection in immune response and immune repair after trauma

Recent studies have reported that miRNA plays an important role in immune response and immune repair after trauma. By regulating the expression of related target genes, miRNA regulates the production, proliferation, development and immune response of immune cells. Therefore, it is in urgent demand to develop an novel method for miRNA analysis. Rolling circle amplification (RCA), as an attractive isothermal signal amplification strategy, has been widely utilized in constructing miRNA detection assays. However, accurate and sensitive miRNA quantitative determination remains a huge challenge for RCA based approaches. Herein, we propose a DSN enzyme based signal cycle initiated Rolling Circle Amplification assay (DiRCA) for sensitive and accurate miRNA detection. In DiRCA, target miRNA unfolds hairpin structure probe in the detection scaffold, forming a RNA–DNA duplex. DSN enzyme is utilized to specifically digest the DNA sequence in RNA–DNA duplex, releasing miRNA to form a signal cycle; its capability to distinguish one base pair mismatch in RNA–DNA duplex endows DiRCA a high selectivity. Meanwhile, DSN enzyme based cleavage initiates RCA, transcribing G-rich sequences for signal generation. Based on the DSN assisted signal cycle and RCA, DiRCA shows a low limit of detection of 0.43 fM and a superior capability in selectively detecting mismatched miRNA sequences, showing a promising prospect in the early-diagnosis of disease.

A universal approach for synthesis of copper nanoclusters templated by G-rich oligonucleotide sequences and their applications in sensing

Herein, five common G4 sequences have been selected, including three different length of telomere DNA, hemin aptamer, and thrombin aptamer, to synthesize Cu nanoclusters (Cu NCs) in-situ. All G4s are proper templates for Cu NCs with low temperature treatment. The particles (G4-Cu NCs) smaller than 3 nm in diameter were obtained and showed light green fluorescence. This is the first report of metal clusters templated by G4s in-situ. As proof of the concept, hemin and alkaline phosphatase (ALP) were used as the targets to test whether the system can monitor the interaction between G4s and its substrate. The results suggest that G4-Cu NCs can indicate the behavior of G4 and its interaction with hemin, and sensing ALP is achieved with the aid of ATP. The linear ranges of hemin and ALP are 300–4000 nM and 10–500 U/L, respectively, and the corresponding limits of detection as low as 97 nM for hemin and 2.8 U/L for ALP. Moreover, this present system has been successfully applied for the detection of ALP in human serum samples with satisfactory recoveries. This synthesis approach is universal, and it can be easily extended to evaluating the formation of G4, or monitoring the interaction between G4 and its substrate, or selective targeting individual G4, or sensitive detection of other important biomarkers by changing template G4 sequence.

Molecular crowding promotes the aggregation of parallel structured G-quadruplexes

G-quadruplexes are widely distributed in cells and are usually essential in mediating biological processes. The intracellular environment is often in a state of molecular crowding, and the current research considerably focuses on the effect of molecular crowding on the conformation of telomeric G-quadruplexes. However, G-quadruplex-forming oligonucleotides are primarily located in the promoter region of the proto-oncogene and on mRNA inside the cell and are reported to fold into parallel structures. Thus, studying the interaction mechanism between ligands and parallel structured G-quadruplexes under crowding conditions is crucial for the design of drugs targeting G-quadruplexes. In our study, molecular crowding was simulated through polyethylene glycol with an average molecular weight of 200 (PEG200) to investigate the parallel structure of the canonical G-quadruplexes c-KIT1, c-MYC, and 32KRAS and their interactions with ligands. Circular dichroism (CD) spectral scanning, fluorescence resonance energy transfer (FRET), and native polyacrylamide gel electrophoresis (PAGE) analysis revealed that molecular crowding failed to induce oligonucleotides to form parallel G-quadruplex structures in the explored model sequences while induced telomeric G-rich sequences to form antiparallel G-quadruplexes in solution without K+. Molecular crowding did not induce changes in their parallel structures but promoted the formation of G-quadruplex aggregates. Moreover, to some extent, molecular crowding also induced a looser structure of the monomer G-quadruplexes. Further studies showed that molecular crowding did not alter the binding stoichiometry of the ligand 3,11-difluoro-6,8,13-trimethyl-8H-quino [4,3,2-kl] acridinium methosulfate (RHPS4) to c-KIT1, while it inhibited its interaction with parallel structured G-quadruplexes. This work provides new insights into developing anticancer drugs targeting parallel structured G-quadruplexes.

G-quadruplex formation at human DAT1 gene promoter: Effect of cytosine methylation

The dopamine transporter gene (DAT1), a recognized genetic risk factor for attention deficit hyperactivity disorder (ADHD) is principally responsible for the regulation of dopamine synaptic levels and serves as a key target in many psychostimulants drugs. DAT1 gene methylation has been considered an epigenetic marker in ADHD. The identification of G-rich sequence motifs potential to form G-quadruplexes is correlated with functionally important genomic regions. Herein, biophysical and biochemical techniques are employed to investigate the structural polymorphism along with the effect of cytosine methylation on a 26-nt G-rich sequence present in the promoter region of the DAT1 gene. The gel electrophoresis, circular dichroism spectroscopy, and UV-thermal melting data are well correlated and conclude the formation of a parallel (bimolecular), as well as antiparallel (tetramolecular) G-quadruplex in Na+ solution. Interestingly, the existence of uni-, bi-, tri-, and tetramolecular quadruplex structures in K+ solution exhibited only the parallel type G-quadruplex. The results demonstrate that in presence of either cation (Na+ or K+) the cytosine methylation reserved the structural topologies unaltered. However, methylation lowers the thermal stability of G-quadruplexes and the duplex structures, as well. These findings provide insights to understand the regulatory mechanisms underlying the formation of the G-quadruplex structure induced by DNA methylation.

A Novel Cu2+ Quantitative Detection Nucleic Acid Biosensors Based on DNAzyme and “Blocker” Beacon

In this paper, a "turn-off" biosensor for detecting copper (II) ions based on Cu2+-dependent DNAzyme and a "blocker" beacon were developed. Upon the copper ion being added, the Cu2+-dependent DNAzyme substrate strand was irreversibly cleaved, thereby blocking the occurrence of the ligation reaction and PCR, which inhibited the G-rich sequence from forming the G-quadruplex structure, efficiently reducing the detection signal. This method had the characteristics of strong specificity and high sensitivity compared with the existing method due to the application of ligation-dependent probe signal recognition and amplification procedures. Under the optimized conditions, this method proved to be highly sensitive. The signal decreased as the concentration of copper ions increased, exhibiting a linear calibration from 0.03125 μM to 0.5 μM and a limit of detection of 18.25 nM. Subsequently, the selectivity of this biosensor was verified to be excellent by testing different relevant metal ions. Furthermore, this detection system of copper (II) ions was successfully applied to monitor Cu2+ contained in actual water samples, which demonstrated the feasibility of the biosensor.

Sensitive and reliable analysis of endometrial cancer related microRNA using ternary hybridization hairpin probe

MicroRNA (miRNA), as a kind of small non-coding ribonucleic acid (RNA) that plays a crucial role in regulating transcriptional activities, is a potential biomarker for EC diagnosis. However, reliable detection of miRNA remains a huge challenge, especially for these methods that require multiple probes for signal amplifications, due to the detective deviation caused by variation of probe concentrations. Herein, we present a novel approach for miRNA-205 identification and quantification by employing simply a ternary hairpin probe (TH probe). The ternary hybridization of three sequences results in the construction of the TH probe, which combines high-efficient signal amplification and specific target recognition. A significant number of G-rich sequences have been produced as a result of the enzymes assisted signal amplification process. The G-rich sequences can fold into G-quadruplexes, which can then be detected in a label-free manner by a common fluorescent dye (thioflavin T). Eventually, the approach exhibits a low limit of detection of 278 aM with a wide detection range of 7 orders of magnitude. In summary, the proposed approach possesses a great potential for both clinical diagnosis of EC and fundamental biomedical researches.

Detection of cancer-associated miRNA using a fluorescence switch of AgNC@NA and guanine-rich overhang sequences.

DNA-templated silver nanoclusters (AgNC@DNA) are a novel type of nanomaterial with advantageous optical properties. Only a few atoms in size, the fluorescence of nanoclusters can be tuned using DNA overhangs. In this study, we explored the properties of AgNCs manufactured on a short single-stranded (dC)12 when adjacent G-rich sequences (dGN , with N = 3-15) were added. The 'red' emission of AgNC@dC12 with λMAX = 660 nm dramatically changed upon the addition of a G-rich overhang with NG = 15. The pattern of the emission-excitation matrix (EEM) suggested the emergence of two new emissive states at λMAX = 575 nm and λMAX = 710 nm. The appearance of these peaks provides an effective way to design biosensors capable of detecting specific nucleic acid sequences with low fluorescence backgrounds. We used this property to construct an NA-based switch that brings AgNC and the G overhang near one another, turning 'ON' the new fluorescence peaks only when a specific miRNA sequence is present. Next, we tested this detection switch on miR-371, which is overexpressed in prostate cancer. The results presented provide evidence that this novel fluorescent switch is both sensitive and specific with a limit of detection close to 22 picomoles of the target miR-371 molecule.

Thymine DNA glycosylase is an RNA-binding protein with high selectivity for G-rich sequences

Thymine DNA glycosylase (TDG) is a multifaceted enzyme involved in several critical biological pathways, including transcriptional activation, DNA demethylation, and DNA repair. Recent studies have established regulatory relationships between TDG and RNA, but the molecular interactions underlying these relationships are poorly understood. Herein, we now demonstrate that TDG binds directly to RNA with nanomolar affinity. Using synthetic oligonucleotides of defined length and sequence, we show that TDG has a strong preference for binding G-rich sequences in single-stranded RNA but binds weakly to single-stranded DNA and duplex RNA. TDG also binds tightly to endogenous RNA sequences. Studies with truncated proteins indicate that TDG binds RNA primarily through its structured catalytic domain and that its disordered C-terminal domain plays a key role in regulating TDG's affinity and selectivity for RNA. Finally, we show that RNA competes with DNA for binding to TDG, resulting in the inhibition of TDG-mediated excision in the presence of RNA. Together, this work provides support for and insights into a mechanism wherein TDG-mediated processes (e.g., DNA demethylation) are regulated through the direct interactions of TDG with RNA.

An efficient DNAzyme-based DNA scaffold for label-free and sensitive bacterial pathogen detection

Even though it is very important, it is still rather difficult to detect minuscule levels of the bacterial pathogen in clinical practice, such as samples from dental implants. We construct here an efficient scaffold for label-free and sensitive Staphylococcus aureus (S. aureus) detection. The precise recognition of target bacteria by the detection scaffold leads to the self-assembly of Chain i and DNAzyme based cleavage of Chain iii. In detail, active DNAzyme conformation is formed based on the hybridization of Chain iii and Chain ii, and a nicking site is generated in Chain iii, making it possible to form a self-primer in Chain i. With the assistance of DNA polymerase, a single-strand DNA chain is added to the 3′ terminal of Chain i, in which process the bacteria is released for the complex to bind with a next detection scaffold, forming a signal recycle. Following DNAzyme-based cleavage, the liberated sequences unroll MB and release G-rich sequences that can specifically bind with the fluorescent dye Thioflavin T (ThT), initiating ThT's fluorescence signal production. The approach demonstrates a wide detection range of 102 CFU/mL and 106 CFU/mL with a low limit of detection of 45 CFU/mL based on the developed detection scaffold, offering good prospects in the diagnosis of bacterial illnesses.

G-Quadruplexes in Repeat Expansion Disorders.

The repeat expansions are the main genetic cause of various neurodegeneration diseases. More than ten kinds of repeat sequences with different lengths, locations, and structures have been confirmed in the past two decades. G-rich repeat sequences, such as CGG and GGGGCC, are reported to form functional G-quadruplexes, participating in many important bioprocesses. In this review, we conducted an overview concerning the contribution of G-quadruplex in repeat expansion disorders and summarized related mechanisms in current pathological studies, including the increasing genetic instabilities in replication and transcription, the toxic RNA foci formed in neurons, and the loss/gain function of proteins and peptides. Furthermore, novel strategies targeting G-quadruplex repeats were developed based on the understanding of disease mechanism. Small molecules and proteins binding to G-quadruplex in repeat expansions were investigated to protect neurons from dysfunction and delay the progression of neurodegeneration. In addition, the effects of environment on the stability of G-quadruplex were discussed, which might be critical factors in the pathological study of repeat expansion disorders.

Roles of G4-DNA and G4-RNA in Class Switch Recombination and Additional Regulations in B-Lymphocytes.

Mature B cells notably diversify immunoglobulin (Ig) production through class switch recombination (CSR), allowing the junction of distant "switch" (S) regions. CSR is initiated by activation-induced deaminase (AID), which targets cytosines adequately exposed within single-stranded DNA of transcribed targeted S regions, with a specific affinity for WRCY motifs. In mammals, G-rich sequences are additionally present in S regions, forming canonical G-quadruplexes (G4s) DNA structures, which favor CSR. Small molecules interacting with G4-DNA (G4 ligands), proved able to regulate CSR in B lymphocytes, either positively (such as for nucleoside diphosphate kinase isoforms) or negatively (such as for RHPS4). G4-DNA is also implicated in the control of transcription, and due to their impact on both CSR and transcriptional regulation, G4-rich sequences likely play a role in the natural history of B cell malignancies. Since G4-DNA stands at multiple locations in the genome, notably within oncogene promoters, it remains to be clarified how it can more specifically promote legitimate CSR in physiology, rather than pathogenic translocation. The specific regulatory role of G4 structures in transcribed DNA and/or in corresponding transcripts and recombination hereby appears as a major issue for understanding immune responses and lymphomagenesis.

Design and Synthesis of Xanthone Analogues Conjugated with Aza-aromatic Substituents as Promising G-Quadruplex Stabilizing Ligands and their Selective Cancer Cell Cytotoxic Action.

We have examined the stabilization of higher-order noncanonical G-quadruplex (G4) DNA structures formed by the G-rich sequences in the promoter region of oncogenes such as c-MYC, c-KIT, VEGF and BCl2 by newly synthesized, novel nitrogen-containing aromatics conjugated to xanthone moiety. Compounds with N-heterocyclic substituents such as pyridine (XNiso), benzimidazole (XBIm), quinoxaline (XQX) and fluorophore dansyl (XDan) showed greater effectiveness in stabilizing the G4 DNA as well as selective cytotoxicity for cancer cells (mainly A549) over normal cells both in terms of UV-Vis spectral titrations and cytotoxicity assay. Both fluorescence spectral titrimetric measurements and circular dichroism (CD) melting experiments further substantiated the G4 stabilization phenomenon by these small-molecular ligands. In addition, these compounds could induce the formation of parallel G4 structures in the absence of any added salt condition in Tris⋅HCl buffer at 25 °C. In a polymerase stop assay, the formation of stable G4 structures in the promoter of oncogenes and halting of DNA synthesis in the presence of the above-mentioned compounds was demonstrated by using oncogene promoter as the DNA synthesis template. Apoptosis-mediated cell death of the cancer cells was proved by Annexin V-PI dual staining assay and cell-cycle arrest occurred in the S phase of the cell cycles. The plausible mode of binding involves the stacking of the xanthone core on the G4 DNA plane with the possibility of interaction with the 5'-overhang as indicated by molecular dynamics simulation studies.

Salphen metal complexes as potential anticancer agents: interaction profile and selectivity studies toward the three G-quadruplex units in the KIT promoter.

DNA G-rich sequences can organize in four-stranded structures called G-quadruplexes (G4s). These motifs are enriched in significant sites within the human genomes, including telomeres and promoters of cancer related genes. For instance, KIT proto-oncogene promoter, associated with diverse cancers, contains three adjacent G4 units, namely Kit2, SP, and Kit1. Aiming at finding new and selective G-quadruplex binders, we have synthesized and characterized five non-charged metal complexes of Pt(II), Pd(II), Ni(II), Cu(II) and Zn(II) of a chlorine substituted Salphen ligand. The crystal structure of the Pt(II) and Pd(II) complexes was determined by XRPD. FRET measurements indicated that Pt(II) and Pd(II) compounds stabilize Kit1 and Kit2 G4s but not SP, telomeric and double stranded DNA. Spectroscopic investigations (UV-Vis, circular dichroism and fluorescence) suggested the Cu(II) complex as the most G4-selective compound. Interestingly, docking simulations indicate that the synthesized compounds fit groove binding pockets of both Kit1 and Kit2 G4s. Moreover, they exhibited dose-dependent cytotoxic activity in MCF-7, HepG2 and HeLa cancer cells.

Resolving a guanine-quadruplex structure in the SARS-CoV-2 genome through circular dichroism and multiscale molecular modeling.

The genome of SARS-CoV-2 coronavirus is made up of a single-stranded RNA fragment that can assume a specific secondary structure, whose stability can influence the virus's ability to reproduce. Recent studies have identified putative guanine quadruplex sequences in SARS-CoV-2 genome fragments that are involved in coding for both structural and non-structural proteins. In this contribution, we focus on a specific G-rich sequence referred to as RG-2, which codes for the non-structural protein 10 (Nsp10) and assumes a guanine-quadruplex (G4) arrangement. We provide the secondary structure of RG-2 G4 at atomistic resolution by molecular modeling and simulation, validated by the superposition of experimental and calculated electronic circular dichroism spectra. Through both experimental and simulation approaches, we have demonstrated that pyridostatin (PDS), a widely recognized G4 binder, can bind to and stabilize RG-2 G4 more strongly than RG-1, another G4 forming sequence that was previously proposed as a potential target for antiviral drug candidates. Overall, this study highlights RG-2 as a valuable target to inhibit the translation and replication of SARS-CoV-2, paving the way towards original therapeutic approaches against emerging RNA viruses.

Anomalies in dye-terminator DNA sequencing caused by a natural G-quadruplex.

A G-rich DNA sequence from yeast that can form a non-canonical G-quadruplex structure was cloned into a plasmid vector and subjected to Sanger sequencing using dye-labeled dideoxynucleotides. Two different effects were observed. In one, presence of the G4 sequence on the template strand led to incorrect incorporation of an A residue at an internal position in the G4 sequence. In the other, the nascent strand caused attenuation of the readout coincident with synthesis of the G-rich DNA. The two effects are novel examples of disruption in DNA synthesis caused by a G4 sequence. These results provide a new example of a DNA structure that could influence genomic stability in human cells.

Novel Chiral Ru(II) Complexes as Potential c-myc G-quadruplex DNA Stabilizers Inducing DNA Damage to Suppress Triple-Negative Breast Cancer Progression.

Currently, effective drugs for triple-negative breast cancer (TNBC) are lacking in clinics. c-myc is one of the core members during TNBC tumorigenesis, and G-rich sequences in the promoter region can form a G-quadruplex conformation, indicating that the c-myc inhibitor is a possible strategy to fight cancer. Herein, a series of chiral ruthenium(II) complexes ([Ru(bpy)2(DPPZ-R)](ClO4)2, Λ/Δ-1: R = -H, Λ/Δ-2: R = -Br, Λ/Δ-3: R = -C≡C(C6H4)NH2) were researched based on their interaction with c-myc G-quadruplex DNA. Λ-3 and Δ-3 show high affinity and stability to decrease their replication. Additional studies showed that Λ-3 and Δ-3 exhibit higher inhibition against different tumor cells than other molecules. Δ-3 decreases the viability of MDA-MB-231 cells with an IC50 of 25.51 μM, which is comparable with that of cisplatin, with an IC50 of 25.9 μM. Moreover, Δ-3 exhibits acceptable cytotoxic activity against MDA-MB-231 cells in a zebrafish xenograft breast cancer model. Further studies suggested that Δ-3 decreases the viability of MDA-MB-231 cells predominantly through DNA-damage-mediated apoptosis, which may be because Δ-3 can induce DNA damage. In summary, the results indicate that Ru(II) complexes containing alkinyl groups can be developed as c-myc G-quadruplex DNA binders to block TNBC progression.

Probing the Tl+-induced structural transformation of G-rich DNA to G-quadruplex with a label‐free thioflavin T-based biosensor

More than 700,000 G-rich sequences in the human genome are able to form G-quadruplex (G4). Since G4 is closely relevant to a variety of essential biological regulatory processes, identification of these G-rich DNAs (G-rich → G4) is thus important for DNA-involved interactions, e.g., metal ions and small molecules. Circular dichroism (CD) and NMR are the dominated tools for studying G4 DNA-based interactions, but suffers from low sensitivity. Herein, with toxic Tl+ as the model inducer, we demonstrated that thioflavin T (ThT), a typical label-free G4 dye, could be explored for highly sensitive probing of Tl+-induced structural transformation of G-rich DNAs to G4. With several model G4-forming DNAs, it was found that the Tl+-induced CD spectra developments correlated well with its fluorescence modulation on ThT (ThT-DNA → ThT-DNA-Tl+). In other words, the Tl+-induced structural change (Free State → G4) could be well probed with the ThT fluorescence change. For typical non-G4-forming DNAs, no appreciable ThT fluorescence modulation was received. To further increase the accuracy of picking up G4-forming DNAs from unknown samples, the photophysical parameters (absorption, fluorescence emission, and fluorescence lifetime) change of ThT before and after Tl+ modulation were integrated to develop a two-step linear discrimination analysis (LDA) protocol, with cross-validation accuracy of over 90%. After picking up G4-forming DNAs from the unknown samples, the Tl+-induced structural transformation of G4 DNAs could be thus evaluated.

Ultrasensitive Isothermal Detection of SARS-CoV-2 Based on Self-Priming Hairpin-Utilized Amplification of the G-Rich Sequence.

The outbreak of the novel coronavirus disease 2019 (COVID-19) pandemic induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused millions of fatalities all over the world. Unquestionably, the effective and timely testing for infected individuals is the most imperative for the prevention of the ongoing pandemic. Herein, a new method was established for detecting SARS-CoV-2 based on the self-priming hairpin-utilized isothermal amplification of the G-rich sequence (SHIAG). In this strategy, the target RNA binding to the hairpin probe (HP) was uniquely devised to lead to the self-priming-mediated extension followed by the continuously repeated nicking and extension reactions, consequently generating abundant G-rich sequences from the intended reaction capable of producing fluorescence signals upon specifically interacting with thioflavin T (ThT). Based on the unique isothermal design concept, we successfully identified SARS-CoV-2 genomic RNA (gRNA) as low as 0.19 fM with excellent selectivity by applying only a single HP and further verified its practical diagnostic capability by reliably testing a total of 100 clinical specimens for COVID-19 with 100% clinical sensitivity and specificity. This study would provide notable insights into the design and evolution of new isothermal strategies for the sensitive and facile detection of SARS-CoV-2 under resource constraints.

A single-stranded DNA-based and terbium (III) fluorescent probe for facile and rapid detection of mercury (II)

Hg2+, as a highly dangerous pollutant, seriously affects public health and food safety. In this work, a biosensor based on single-stranded DNA and Tb3+ fluorescent probe was constructed for facile and rapid detection of Hg2+. The detection method only needs one DNA strand, which consists of two parts: the G-rich sequence is the Tb3+ luminescent antenna ligand and the T-rich sequence is the Hg2+ recognition probe. Free mercury-specific DNA (MSD) in an aqueous solution could significantly enhance Tb3+ luminescence. Upon the addition of Hg2+, the conformation of MSD will switch from the free random coil structure to the hairpin structure. Thus, Tb3+ was released and the fluorescence of Tb3+ was decreased. After condition optimization, the change of fluorescence intensity was proportional to the logarithmic concentration of Hg2+ from 0.01 to 10 μM with a detection limit of 1.9 nM. In addition, the detection results of Hg2+ in real samples were satisfactory. The designed fluorescent probe has great advantages in simplicity and functionality and has great development potential in food safety and environmental pollution.