Human Telomere Research Articles

A structure-function approach to elucidate the molecular mechanism of the telomere C-strand fill-in process

Telomeres are repetitive DNA sequences decorated with telomere-specific proteins at the ends of linear chromosomes in eukaryotic cells. The regulation of human telomere length is essential for maintaining genome stability and cellular lifespan, and its dysregulation causes premature aging and cancer. The heterotrimeric CTC1-STN1-TEN1 (CST) protein complex is vital for the timely termination of telomere elongation by telomerase. CST is also required to recruit DNA polymerase α-primase (pol α-primase) to the newly synthesized telomeric G-overhang ssDNA to convert the telomeric ssDNA to dsDNA (known as C-strand fill-in).

Cyclic ferrocenylnaphthalene diimides as a probe for electrochemical telomerase assay

Novel cyclic naphthalene diimides, 8 and 12, containing ferrocene in the cyclic linker were synthesized as G-quartet (G4) specific electrochemical ligands via the reaction of 1,1′-ferrocenedipropanoic acid and the terminal amine moieties of naphthalene diimides with varying linker lengths. The redox potentials of 8 and 12 were ca. 0.2 V (vs. Ag/AgCl), and the background current in an electrolyte was successfully suppressed. Both 8 and 12 bound to TA-core, representing human telomere G4, with K = 4.4 and 38 × 105 M−1, respectively. The current response of 12 to an electrode immobilized with G4 was the highest among the acyclic derivatives, suggesting its potential application in electrochemical telomerase assays.

Computational and Experimental Characterization of rDNA and rRNA G-Quadruplexes.

DNA G-quadruplexes in human telomeres and gene promoters are being extensively studied for their role in controlling the growth of cancer cells. G-quadruplexes have been unambiguously shown to exist both in vitro and in vivo, including in the guanine (G)-rich DNA genes encoding pre-ribosomal RNA (pre-rRNA), which is transcribed in the cell's nucleolus. Recent studies strongly suggest that these DNA sequences ("rDNA"), and the transcribed rRNA, are a potential anticancer target through the inhibition of RNA polymerase I (Pol I) in ribosome biogenesis, but the structures of ribosomal G-quadruplexes at atomic resolution are unknown and very little biophysical characterization has been performed on them to date. In the present study, circular dichroism (CD) spectroscopy is used to show that two putative rDNA G-quadruplex sequences, NUC 19P and NUC 23P and their counterpart rRNAs, predominantly adopt parallel topologies, reminiscent of the analogous telomeric quadruplex structures. Based on this information, we modeled parallel topology atomistic structures of the putative ribosomal G-quadruplexes. We then validated and refined the modeled ribosomal G-quadruplex structures using all-atom molecular dynamics (MD) simulations with the CHARMM36 force field in the presence and absence of stabilizing K+. Motivated by preliminary MD simulations of the telomeric parallel G-quadruplex (TEL 24P) in which the K+ ion is expelled, we used updated CHARMM36 force field K+ parameters that were optimized, targeting the data from quantum mechanical calculations and the polarizable Drude model force field. In subsequent MD simulations with optimized CHARMM36 parameters, the K+ ions are predominantly in the G-quadruplex channel and the rDNA G-quadruplexes have more well-defined, predominantly parallel-topology structures as compared to rRNA. In addition, NUC 19P is more structured than NUC 23P, which contains extended loops. Results from this study set the structural foundation for understanding G-quadruplex functions and the design of novel chemotherapeutics against these nucleolar targets and can be readily extended to other DNA and RNA G-quadruplexes.

Prenatal PM2.5 Exposure in Relation to Maternal and Newborn Telomere Length at Delivery.

Particulate matter with an aerodynamic diameter of 2.5 μm or less (PM2.5) is a ubiquitous air pollutant that is increasingly threatening the health of adults and children worldwide. One health impact of elevated PM2.5 exposure is alterations in telomere length (TL)—protective caps on chromosome ends that shorten with each cell division. Few analyses involve prenatal PM2.5 exposure, and paired maternal and cord TL measurements. Here, we analyzed the association between average and trimester-specific prenatal PM2.5 exposure, and maternal and newborn relative leukocyte TL measured at birth among 193 mothers and their newborns enrolled in a New-York-City-based birth cohort. Results indicated an overall negative relationship between prenatal PM2.5 and maternal TL at delivery, with a significant association observed in the second trimester (β = −0.039, 95% CI: −0.074, −0.003). PM2.5 exposure in trimester two was also inversely related to cord TL; however, this result did not reach statistical significance (β = −0.037, 95% CI: −0.114, 0.039), and no clear pattern emerged between PM2.5 and cord TL across the different exposure periods. Our analysis contributes to a limited body of research on ambient air pollution and human telomeres, and emphasizes the need for continued investigation into how PM2.5 exposure during pregnancy influences maternal and newborn health.

Detection of TERRA R-Loops at Human Telomeres.

R-loops are three-stranded nucleic acid structures composed of a DNA-RNA hybrid and a displaced DNA strand. The long noncoding RNA TERRA forms R-loops at telomeres influencing the telomeric chromatin composition and impacting on telomere maintenance mechanisms by semiconservative DNA replication, homology directed DNA repair and telomerase. Here, we describe a method to detect R-loops at telomeres, which involves immunoprecipitation with the R-loop recognizing S9.6 antibody, followed by detection of telomeric DNA by either dot-blot hybridization with a radiolabeled telomeric probe, or qPCR using DNA primers that are specific for subtelomeric sequences.

The Use of Ruthenium Complexes as Molecular Probes for Non-Canonical DNA

This study considered the preparation of a new DNA binding Ruthenium polypyridyl complex possessing an infrared active nitrile group. The binding abilities of a novel Ruthenium complex, [Ru(TMP)2DPPZ-10-CN], to various forms of DNA—both canonical and non-canonical—were examined by performing multiple DNA titrations. DNA is of great interest as it is the carrier of genetic information for all living things. Damage to DNA can have drastically detrimental effects, so the study of its structure and replication is of great importance. Two non-canonical structures that are important are the G-quadruplex and i-motif which form at the telomeric and regulatory regions of genes, respectively, and have the ability to block telomerase activity and influence transcription. The complex was synthesized by microwave irradiation and purified using a silica column and an ion exchange with Amberlite 402. Six titrations were, then, performed with salmon sperm dsDNA, guanine monophosphate (GMP), G4T4G4, human telomere G-quadruplex, i-motif C5T3, and i-motif C30. The complex was found to favor non-canonical structures, particularly the G-quadruplex structure, because of its high [bp]/[Ru] concentrations. The higher concentration of base pairs or structures per Ruthenium molecule indicated that the complex had a high binding affinity for that particular DNA structure. These results support the notion that Ruthenium metal complexes can be used for theragnostic purposes and can be used to target the telomeric region of genes where G-quadruplex structures can be found and influence transcription initiation and inhibit telomerase activity.

The Use of Ruthenium Complexes as Molecular Probes for Non-Canonical DNA” by Eleni Sullivan

This study considered the preparation of a new DNA binding Ruthenium polypyridyl complex possessing an infrared active nitrile group. The binding abilities of a novel Ruthenium complex, [Ru(TMP)2DPPZ-10-CN], to various forms of DNA—both canonical and non-canonical—were examined by performing multiple DNA titrations. DNA is of great interest as it is the carrier of genetic information for all living things. Damage to DNA can have drastically detrimental effects, so the study of its structure and replication is of great importance. Two non-canonical structures that are important are the G-quadruplex and i-motif which form at the telomeric and regulatory regions of genes, respectively, and have the ability to block telomerase activity and influence transcription. The complex was synthesized by microwave irradiation and purified using a silica column and an ion exchange with Amberlite 402. Six titrations were, then, performed with salmon sperm dsDNA, guanine monophosphate (GMP), G4T4G4, human telomere G-quadruplex, i-motif C5T3, and i-motif C30. The complex was found to favor non-canonical structures, particularly the G-quadruplex structure, because of its high [bp]/[Ru] concentrations. The higher concentration of base pairs or structures per Ruthenium molecule indicated that the complex had a high binding affinity for that particular DNA structure. These results support the notion that Ruthenium metal complexes can be used for theragnostic purposes and can be used to target the telomeric region of genes where G-quadruplex structures can be found and influence transcription initiation and inhibit telomerase activity.

Telomeres, aging, and cancer: the big picture

AbstractThe role of telomeres in human health and disease is yet to be fully understood. The limitations of mouse models for the study of human telomere biology and difficulties in accurately measuring the length of telomere repeats in chromosomes and cells have diverted attention from many important and relevant observations. The goal of this perspective is to summarize some of these observations and to discuss the antagonistic role of telomere loss in aging and cancer in the context of developmental biology, cell turnover, and evolution. It is proposed that both damage to DNA and replicative loss of telomeric DNA contribute to aging in humans, with the differences in leukocyte telomere length between humans being linked to the risk of developing specific diseases. These ideas are captured in the Telomere Erosion in Disposable Soma theory of aging proposed herein.

Age-Related Phenotypes Linked to Aberrant Expression and Localization of a Telomeric Protein

Telomere attrition is associated with telomere biology disorders and age-related diseases. In telomere biology disorders, telomere uncapping induces a DNA damage response that evokes cell death or senescence. However, a causal mechanism for telomere attrition in age-related diseases remains elusive. Telomere capping and integrity are maintained by shelterin, a six-protein complex. Rap1 is the only shelterin member that is not required for telomere capping and is expressed at non-telomeric genomic and cytosolic regions. The objective of this study was to determine aberrant phenotypes attributed to non-telomeric Rap1. To test this, we generated a Rap1 mutant knock-in (KI) mouse model using CRISPR/Cas9 editing, in which Rap1 at telomeres is prevented, leaving only non-telomeric Rap1. Cell fractionation/western blotting of primary fibroblasts from Rap1 KI mice demonstrated decreased Rap1 expression and Rap1 re-localization off telomeres, with an altered cellular distribution. This same difference in Rap1 is also observed in human cells with telomere erosion, indicating that aberrant Rap1 in our model may recapitulate Rap1 in aging and human telomere biology disorders. Compared to wild-type control mice, Rap1 KI mice exhibited increased body weight, altered cytokine levels, behavioral deficits, and decreased lifespan. In conclusion, our results reveal a novel mechanism by which telomere shortening may contribute to age-related pathologies by disrupting Rap1 expression and cell localization.

TRF2 promotes dynamic and stepwise looping of POT1 bound telomeric overhang.

Human telomeres are protected by shelterin proteins, but how telomeres maintain a dynamic structure remains elusive. Here, we report an unexpected activity of POT1 in imparting conformational dynamics of the telomere overhang, even at a monomer level. Strikingly, such POT1-induced overhang dynamics is greatly enhanced when TRF2 engages with the telomere duplex. Interestingly, TRF2, but not TRF2ΔB, recruits POT1-bound overhangs to the telomere ds/ss junction and induces a discrete stepwise movement up and down the axis of telomere duplex. The same steps are observed regardless of the length of the POT1-bound overhang, suggesting a tightly regulated conformational dynamic coordinated by TRF2 and POT1. TPP1 and TIN2 which physically connect POT1 and TRF2 act to generate a smooth movement along the axis of the telomere duplex. Our results suggest a plausible mechanism wherein telomeres maintain a dynamic structure orchestrated by shelterin.

Pressure Perturbation Studies of Noncanonical Viral Nucleic Acid Structures.

Simple SummaryIt is well known that nucleic acids adopt a double-helical structure that is essential in the storage and replication of genetic code. However, noncanonical structures of DNA are less explored. The most important of these structures are the G-quadruplexes, which are formed by guanine-rich sequences of the genome. These G-quadruplexes were found in several crucial positions of the genome; they regulate important processes such as cell proliferation and death. Here we are investigating the quadruplex structures appearing in the genome of the hepatitis B, whose infection is among the ten leading causes of death. Our unique approach—the high-pressure technique—allowed us to characterize the stability of these quadruplexes in wide temperature and pressure ranges. Pressure experiments gave us the volume changes occurring at the unfolding, e.g., the embedded volume of the folded structure. Volumetric parameters are especially important because the space available for molecules is quite limited in the crowded environment of the cell. We also investigated how the stability of the viral quadruplexes can be increased by the binding of TMPyP4, a special ligand developed for cancer therapy.G-quadruplexes are noncanonical structures formed by guanine-rich sequences of the genome. They are found in crucial loci of the human genome, they take part in the regulation of important processes like cell proliferation and cell death. Much less is known about the subjects of this work, the viral G-quadruplexes. We have chosen three potentially G-quadruplex-forming sequences of hepatitis B. We measured the stability and the thermodynamic parameters of these quadruplexes. We also investigated the potential stabilization of these G-quadruplexes by binding a special ligand that was originally developed for cancer therapy. Fluorescence and infrared spectroscopic measurements were performed over wide temperature and pressure ranges. Our experiments indicate the small unfolding volume change of all three oligos. We found a difference between the unfolding of the 2-quartet and the 3-quartet G-quadruplexes. All three G-quadruplexes were stabilized by TMPyP4, which is a cationic porphyrin developed for stabilizing the human telomere.

Molecular insights into the selective binding mechanism targeting parallel human telomeric G-quadruplex

Stabilizing human telomere DNA G-quadruplex (G4) proves a promising anti-cancer strategy. Though plenty of G4 stabilizing molecules have been reported, little is known about their selective binding mechanism among various G4s. Recently, a designed monohydrazone derivative (compound 15) was reported to display specific preference in binding and stabilizing parallel human telomeric G4. To reveal the selective binding mechanism, a comparative theoretical investigation was performed on two monohydrazone derivatives (compounds 1 and 15) and three telomeric G4s showing parallel, hybrid-I, and hybrid-II conformations. Two probable binding modes, i.e. the end-stacking binding and the groove binding, were predicted by molecular dockings for each monohydrazone in its binding with the telomeric G4s. Further long-timescale molecular dynamics simulations reveal the conversion from the groove binding to the end-stacking binding for both compounds, indicating the preference of the end-stacking binding mode. Structural analysis together with binding free energy calculations show that the van der Waals interaction plays a leading role in ranking the binding affinity. By forming extensive van der Waals interactions, the parallel G4–15 binding complex shows the highest binding affinity, and the corresponding compound 15 exhibits the strongest stabilizing effect to the telomeric G4. These findings agree well with the experimental observations. Through characterizing the selective binding between monohydrazones and telomeric G4s at the atomic level, the current study provides support to the design of novel selective stabilizers targeting telomeric G4s.

A Quantitative Proteomic Approach for the Identification of DNA Guanine Quadruplex-Binding Proteins.

DNA sequences of high guanine (G) content have the potential to form G quadruplex (G4) structures. A more complete understanding about the biological functions of G4 DNA requires the investigation about how these structures are recognized by proteins. Here, we conducted exhaustive quantitative proteomic experiments to profile the interaction proteomes of G4 structures by employing different sequences of G4 DNA derived from the human telomere and the promoters of c-MYC and c-KIT genes. Our results led to the identification of a number of candidate G4-interacting proteins, many of which were discovered here for the first time. These included three proteins that can bind to all three DNA G4 structures and 78 other proteins that can bind selectively to one or two of the three DNA G4 structure(s). We also validated that GRSF1 can bind directly and selectively toward G4 structure derived from the c-MYC promoter. Our quantitative proteomic screening also led to the identification of a number of candidate "antireader" proteins of G4 DNA. Together, we uncovered a number of cellular proteins that exhibit general and selective recognitions of G4 folding patterns, which underscore the complexity of G4 DNA in biology and the importance of understanding fully the G4-interaction proteome.

Chemical Modulation of DNA Replication along G-Quadruplex Based on Topology-Dependent Ligand Binding.

Ligands that bind to and stabilize guanine-quadruplex (G4) structures to regulate DNA replication have therapeutic potential for cancer and neurodegenerative diseases. Because there are several G4 topologies, ligands that bind to their specific types may have the ability to preferentially regulate the replication of only certain genes. Here, we demonstrated that binding ligands stalled the replication of template DNA at G4, depending on different topologies. For example, naphthalene diimide derivatives bound to the G-quartet of G4 with an additional interaction between the ligand and the loop region of a hybrid G4 type from human telomeres, which efficiently repressed the replication of the G4. Thus, these inhibitory effects were not only stability-dependent but also topology-selective based on the manner in which G4 structures interacted with G4 ligands. Our original method, referred to as a quantitative study of topology-dependent replication (QSTR), was developed to evaluate correlations between replication rate and G4 stability. QSTR enabled the systematic categorization of ligands based on topology-dependent binding. It also demonstrated accuracy in determining quantitatively how G4 ligands control the intermediate state of replication and the kinetics of G4 unwinding. Hence, the QSTR index would facilitate the design of new drugs capable of controlling the topology-dependent regulation of gene expression.

Multiple hPOT1-TPP1 cooperatively unfold contiguous telomeric G-quadruplexes proceeding from 3' toward 5', a feature due to a 3'-end binding preference and to structuring of telomeric DNA.

Telomeres are DNA repeated sequences that associate with shelterin proteins and protect the ends of eukaryotic chromosomes. Human telomeres are composed of 5′TTAGGG repeats and ends with a 3′ single-stranded tail, called G-overhang, that can be specifically bound by the shelterin protein hPOT1 (human Protection of Telomeres 1). In vitro studies have shown that the telomeric G-strand can fold into stable contiguous G-quadruplexes (G4). In the present study we investigated how hPOT1, in complex with its shelterin partner TPP1, binds to telomeric sequences structured into contiguous G4 in potassium solutions. We observed that binding of multiple hPOT1–TPP1 preferentially proceeds from 3′ toward 5′. We explain this directionality in terms of two factors: (i) the preference of hPOT1–TPP1 for the binding site situated at the 3′ end of a telomeric sequence and (ii) the cooperative binding displayed by hPOT1–TPP1 in potassium. By comparing binding in K+ and in Li+, we demonstrate that this cooperative behaviour does not stem from protein-protein interactions, but from structuring of the telomeric DNA substrate into contiguous G4 in potassium. Our study suggests that POT1-TPP1, in physiological conditions, might preferentially cover the telomeric G-overhang starting from the 3′-end and proceeding toward 5′.

Association between maternal urinary selenium during pregnancy and newborn telomere length: results from a birth cohort study.

Newborn telomere length is considered as an effective predictor of lifespan and health outcomes in later life. Selenium is an essential trace element for human health, and its antioxidation is of great significance for the prevention of telomere erosion. We recruited 746 mother-newborn pairs in Wuhan Children's Hospital between 2013 and 2015. Urine samples were repeatedly collected at three time points during pregnancy, and umbilical cord blood samples were collected right after parturition. Urinary selenium concentration was detected using inductively coupled plasma mass spectrometry, and newborn telomere length was measured using quantitative real-time polymerase chain reaction. We applied general estimating equations to examine the trimester-specific association between maternal urinary selenium during pregnancy and newborn telomere length. The median of creatinine-corrected selenium concentrations during pregnancy were 16.29, 18.08, and 18.35 μg/g·creatinine in the first, second, and third trimesters, respectively. Selenium concentrations in all the three trimesters were significantly associated with newborn telomere length. Per doubling of maternal urinary selenium concentrations was associated with 6.44% (95% CI: 0.92, 12.25), 6.54% (95% CI: 0.17, 13.31), and 6.02% (95% CI: 0.29, 12.09) longer newborn telomere length in the first, second, and third trimesters, respectively, after adjusting for potential confounders. This is the first study to provide evidence for the effect of maternal selenium levels on fetal telomere erosion. Findings from our study suggested that maternal urinary selenium was positively associated with newborn telomere length, indicating that intrauterine selenium exposure might have effect on initial setting of human telomere length.

Investigation on the competition of duplex/ G-quadruplex/ i-motif in telomere sequences and c-MYC gene with a solid-state nanopore sensor

Apart from duplex helix, the coexisted noncanonical DNA may be involved in inhibiting DNA replication, and controlling gene expression. The competition of duplex/ G4/ i-motif depends on many factors, including sequence information, external conditions and binding effects. This work looked into the human telomere sequences of different length (hTelo-1, 2) and intramolecular G-rich and C-rich strands (hTelo-3, 4, 5). Nanopore translocation properties suggested that in the presence of their complementary sequences in the mixed K+/Cs+ buffer, G4 was the major species, and G4 from hTelo-2 that holds multiple G-quartets formed slower than shorter hTelo-1. The intramolecular G/C-rich strands hTelo-3, 4, 5 predominantly formed duplex rather than quadruplex under K+ or acidic conditions, indicating that intramolecular quadruplex is less stable than duplex due to the strand strain. Translocation behaviors of mixed proto-oncogene cMYC/c-cMYC illustrated that G4 was formed in both K+ and Cs+, and Cs+ inhibited the duplex formation in K+/Cs+ buffer. The increase of the ratio of Cs+ will decrease the duplexes species. Our work demonstrated the relative stability of duplex/quadruplex under various conditions with distinct strands, which is useful for the investigation of DNA secondary structural dynamics and transition mechanism in biological function regions, and paved the way for the potential of disease-targeted diagnosis.

The different biological effects of TMPyP4 and cisplatin in the inflammatory microenvironment of osteosarcoma are attributed to G-quadruplex.

ObjectiveOsteosarcoma (OS) is characterized by high levels of the tumour‐associated inflammatory microenvironment. Moreover, in approximately 60% of OS, telomere length is maintained by alternative lengthening of telomeres (ALT) pathway. Whether the ALT pathway can be exploited for OS therapeutic treatment and how the OS inflammatory microenvironment influences the anti‐cancer drug effect remains unknown. Here, we examined the biological effects of TMPyP4 and cisplatin in the inflammatory microenvironment of OS cells.Materials and methodsImmunofluorescence in situ hybridization (IF‐FISH) and C‐circle experiments were used to detect the G‐quadruplex and ALT activity. The redox potential of single guanine, G‐quadruplex and G‐quadruplex/TMPyP4 was evaluated by the lowest unoccupied molecular orbital energy (LUMO), zeta potential and cyclic voltammetry. Cell viability, flow cytometry and apoptosis, Western blot, comet assay, adhesion, transwell and scratch experiments were performed to compare the anti‐tumour proliferation and migration effects of TMPyP4 and cisplatin in the inflammatory microenvironment.ResultsThis study indicated that compared with cisplatin, TMPyP4 could induce the formation of human telomeres and FAK G‐quadruplex in vitro and in vivo, and TMPyP4‐treated OS cells showed fewer extrachromosomal C‐circles and fewer ALT‐associated promyelocytic leukaemia bodies. Consequently, the ALT activity and FAK‐related cell migration were suppressed by TMPyP4. Mechanistically, the formation of G‐quadruplex resulted in both lower redox potential than G within the genome and FAK transcription inhibition, and TMPyP4 could enhance this phenomenon, especially in the inflammatory microenvironment.ConclusionsOur results reveal that TMPyP4 is more suitable for OS treatment than cisplatin.

New (Iso)quinolinyl-pyridine-2,6-dicarboxamide G-Quadruplex Stabilizers. A Structure-Activity Relationship Study.

G-quadruplex (G4)-interactive small molecules have a wide range of potential applications, not only as drugs, but also as sensors of quadruplex structures. The purpose of this work is the synthesis of analogues of the bis-methylquinolinium-pyridine-2,6-dicarboxamide G4 ligand 360A, to identify relevant structure–activity relationships to apply to the design of other G4-interactive small molecules bearing bis-quinoline or bis-isoquinoline moieties. Thermal denaturation experiments revealed that non-methylated derivatives with a relative 1,4 position between the amide linker and the nitrogen of the quinoline ring are moderate G4 stabilizers, with a preference for the hybrid h-Telo G4, a 21-nt sequence present in human telomeres. Insertion of a positive charge upon methylation of quinoline/isoquinoline nitrogen increases compounds’ ability to selectively stabilize G4s compared to duplex DNA, with a preference for parallel structures. Among these, compounds having a relative 1,3-position between the charged methylquinolinium/isoquinolinium nitrogen and the amide linker are the best G4 stabilizers. More interestingly, these ligands showed different capacities to selectively block DNA polymerization in a PCR-stop assay and to induce G4 conformation switches of hybrid h-Telo G4. Molecular dynamic simulations with the parallel G4 formed by a 21-nt sequence present in k-RAS gene promoter, showed that the relative spatial orientation of the two methylated quinoline/isoquinoline rings determines the ligands mode and strength of binding to G4s.

Near-Infrared Small Molecule as a Specific Fluorescent Probe for Ultrasensitive Recognition of Antiparallel Human Telomere G-Quadruplexes.

In the past 10 years, many fluorescent probes have been developed to recognize G-quadruplexes (G4s) since G4s play an important role in biological systems. However, the selectivity and sensitivity of existing probes for G4s limit their further applications. Herein, we design and synthesize a new probe (TOVJ) by introducing 9-vinyljulolidine into TO. The new probe exhibits almost no fluorescence in an aqueous solution. Upon interacting with G4s, especially the antiparallel G4s, the fluorescence intensity was greatly enhanced (maximum 2742-fold) with a large Stokes shift of 198 nm and the maximum emission peak at 694 nm (near-infrared region). TOVJ showed high sensitivity and selectivity to G4s over other DNA topologies (ssDNA/dsDNA), especially to antiparallel G4s. For antiparallel human telomere G4 detection, the limits of detection of Hum24 and 22AG Na+ were as low as 164 and 231 pM, respectively. This indicates that TOVJ is a highly sensitive fluorescence sensor that can be effectively used for antiparallel human telomere G4 detection. The result of live-cell imaging showed that TOVJ could enter live cells and locate in the mitochondria.