The influence of the G‐quartet structural integrity on the catalytic activity of the G‐quadruplex(G4)was investigated by comparing the G4‐DNAzyme performances of a series of G4s with a G‐vacancy site and a G‐trip...The influence of the G‐quartet structural integrity on the catalytic activity of the G‐quadruplex(G4)was investigated by comparing the G4‐DNAzyme performances of a series of G4s with a G‐vacancy site and a G‐triplex(G‐tri).The results presented herein not only confirm that the structural integrity of the 3'‐end G‐quartet is necessary for G4s to be catalytically competent but also show how to remediate G‐vacancy‐mediated catalytic activity losses via the addition of guanine surrogates in an approach referred to as G‐vacancy complementation strategy that is applicable to parallel G4s only.Furthermore,this study demonstrates that the terminal G‐quartet could act as a proximal coordinating group and cooperate with the flanking nucleotide to activate the hemin cofactor.展开更多
Tomato brown rugose fruit virus(ToBRFV) is a novel tobamovirus firstly reported in 2015 and poses a severe threat to the tomato industry. So far, it has spread to 10 countries in America, Asia, and Europe. In 2019, To...Tomato brown rugose fruit virus(ToBRFV) is a novel tobamovirus firstly reported in 2015 and poses a severe threat to the tomato industry. So far, it has spread to 10 countries in America, Asia, and Europe. In 2019, ToBRFV was identified in Shandong Province(ToBRFV-SD), China. In this study, it was shown that ToBRFV-SD induced mild to severe mosaic and blistering on leaves, necrosis on sepals and pedicles, and deformation, yellow spots, and brown rugose necrotic lesions on fruits. ToBRFV-SD induced distinct symptoms on plants of tomato, Capsicum annumm, and Nicotiana benthamiana, and caused latent infection on plants of Solanum tuberosum, Solanum melongena, and N. tabacum cv. Zhongyan 102. All the 50 tomato cultivars tested were highly sensitive to ToBRFV-SD. The complete genomic sequence of ToBRFV-SD shared the highest nucleotide and amino acid identities with isolate IL from Israel. In the phylogenetic tree constructed with the complete genomic sequence, all the ToBRFV isolates were clustered together and formed a sister branch with tobacco mosaic virus(TMV). Furthermore, a quadruplex RT-PCR system was developed that could differentiate ToBRFV from other economically important viruses affecting tomatoes, such as TMV, tomato mosaic virus, and tomato spotted wilt virus. The findings of this study enhance our understanding of the biological and molecular characteristics of ToBRFV and provide an efficient and effective detection method for multiple infections, which is helpful in the management of ToBRFV.展开更多
The G-quadruplex （G4） elements comprise a class of nucleic acid structures formed by stacking of guanine base quartets in a quadruple helix. This （34 DNA can form within or across single-stranded DNA molecules and ...The G-quadruplex （G4） elements comprise a class of nucleic acid structures formed by stacking of guanine base quartets in a quadruple helix. This （34 DNA can form within or across single-stranded DNA molecules and is mutually exclusive with duplex B-form DNA. The reversibility and structural diversity of G4s make them highly versatile genetic structures, as demonstrated by their roles in various functions including telomere metabolism, genome maintenance, immunoglobulin gene diversification, transcription, and translation. Sequence motifs capable of forming G4 DNA are typically located in telomere repeat DNA and other non-telomeric genomic loci. To investigate their potential roles in a large-genome model plant species, we computationaily identified 149,988 non-telomeric G4 motifs in maize （Zea mays L., B73 AGPv2）, 29% of which were in non-repetitive genomic regions. G4 motif hotspots exhibited non-random enrichment in genes at two locations on the antisense strand, one in the 5~ UTR and the other at the 5~ end of the first intron. Several genic G4 motifs were shown to adopt sequence-specific and potassium-dependent G4 DNA structures in vitro. The G4 motifs were prevalent in key regulatory genes associated with hypoxia （group VII ERFs）, oxidative stress （D J-1/GATasel）, and energy status （AMPK/ SnRK） pathways. They also showed statistical enrichment for genes in metabolic pathways that function in glycolysis, sugar degradation, inositol metabolism, and base excision repair. Collectively, the maize G4 motifs may represent conditional regulatory elements that can aid in energy status gene responses. Such a network of elements could provide a mechanistic basis for linking energy status signals to gene regulation in maize, a model genetic system and major world crop species for feed, food, and fuel.展开更多
In this assay, a label-free fluorescent sensing platform based on triple-helix molecular switch（THMS） and G-quadruplex was developed for the detection of tetracycline. We demonstrated this approach by using THMS, wh...In this assay, a label-free fluorescent sensing platform based on triple-helix molecular switch（THMS） and G-quadruplex was developed for the detection of tetracycline. We demonstrated this approach by using THMS, which consists of a central section with a shortened 8-mer aptamer sequence with high affinity to tetracycline and flanked by two arm segments. G-rich oligonucleotide can specifically bind to thioflavin T（Th T） as a signal transduction probe（STP）. In the absence of tetracycline, THMS remains stable, the fluorescence of background is low. By the addition of target tetracycline, the aptamer-target binding results in the formation of a structured aptamer-target complex, which disassembles the THMS and releases the STP. The free STP self-assembles into G-quadruplex and specifically binds to Th T which generates a obvious fluorescence enhancement. Using the triple-helix molecular switch, the developed aptamer-based fluorescent sensing platform showed a linear relationship with the concentration of tetracycline ranging from 0.2 to 20.0 nmol/L. The detection limit of tetracycline was determined to be970.0 pmol/L. The assay avoids complicated modifications or chemical labeling, making it simple and cost-effective. So, it is expected that this aptamer-based fluorescent assay could be extensively applied in the field of food safety inspection.展开更多
The G-quadruplexes formed from G-rich strands in the telomere and oncogene-promoter regions are regarded as new promising targets in the cancer therapy.A G-quadruplex in the downstream flanking region of the signal tr...The G-quadruplexes formed from G-rich strands in the telomere and oncogene-promoter regions are regarded as new promising targets in the cancer therapy.A G-quadruplex in the downstream flanking region of the signal transducer and activator of transcription 3（STAT3） gene was explored.Its folding patterns were proposed to be 3：2：2 and 3：3：1 loop isomers by the mutation analysis by CD spectroscopy.The structures were constructed and refined by molecular modeling method.展开更多
In this research, an unusually dimeric G-quadruplex of d（GGGTGGGTGGGTGGGT） （SI）, the potent nanomolar HIV-1 integrase inhibitor, was detected by nuclear magnetic resonance （NMR）. This result has been confirmed b...In this research, an unusually dimeric G-quadruplex of d（GGGTGGGTGGGTGGGT） （SI）, the potent nanomolar HIV-1 integrase inhibitor, was detected by nuclear magnetic resonance （NMR）. This result has been confirmed by electrospray ionization mass spectrometry （ESI-MS） and circular dichroism （CD）.展开更多
G-quadruplexes （G4s） play important roles in biological systems, such as telomere maintenance, replication, and transcription. Based on the DNA sequence, loop geometry, and the local environments, G4s can be classif...G-quadruplexes （G4s） play important roles in biological systems, such as telomere maintenance, replication, and transcription. Based on the DNA sequence, loop geometry, and the local environments, G4s can be classified into different conformations. It is important to detect different types of G4s to monitor the diseases related with G4s. Most ligands bind to G4s based on end-stacking modes, while rare ligands bind to G4s through groove binding modes. We have found that a cyanine dye DMSB interacts with parallel G4 by end-stacking and groove simultaneous binding mode. In this article, we found that DMSB could simply discriminate parallel G4s from other DNA motifs by using UV-vis spectrum. These results give some clues to develop high specificity G4 probes.展开更多
G-quadruplex ligands have been accepted as potential therapeutic agents for anticancer treatment. Thioflavin T （ThT）, a highly selective G-quadruplex ligand, can bind G-quadruplex with a fluorescent light-up signal ...G-quadruplex ligands have been accepted as potential therapeutic agents for anticancer treatment. Thioflavin T （ThT）, a highly selective G-quadruplex ligand, can bind G-quadruplex with a fluorescent light-up signal change and high specificity against DNA duplex. However, there are still different opinions that ThT induces/stabilizes G-quadruplex foldings/topologies in human telomere sequence. Here, a sensitive single-molecule nanopore technology was utilized to analyze the interactions between human telomeric DNA （Tel DNA） and ThT. Both translocation time and current blockade were measured to investigate the translocation behaviors. Furthermore, the effects of metal ion （K~ and Na~） and pH on the translocation behaviors were studied. Based on the single-molecule level analysis, there are some conclusions： （1） In the electrolyte solution containing 50 mmol/L I（Cl and 450 mmol/L NaCl, ThT can bind strongly with Tel DNA but nearly does not change the G-quadruplex form; （2） in the presence of high concentration K~, the ThT binding induces the structural change of hybrid G-quadruplex into antiparallel topology with an enhanced structural stability; （3） In either alkaline or acidic buffer, G-quadruplex form will change in certain degree. All above conclusions are helpful to deeply understand the interaction behaviors between Tel DNA and ThT. This nanopore platform, investigating G-quadruplex ligands at the single-molecule level, has great potential for the design of new drugs and sensors.展开更多
The DNA G quadruplex formed by the human telomeric sequence is a potential target for novel anticancer drugs. We have investigated an intramolecular DNA G quadruplex using single molecule fluorescence resonance energy...The DNA G quadruplex formed by the human telomeric sequence is a potential target for novel anticancer drugs. We have investigated an intramolecular DNA G quadruplex using single molecule fluorescence resonance energy transfer and shown that individual folded quadruplexes can be identified. The mean proximity ratio measured at the single molecule level was consistent with ensemble measurement展开更多
The Bloom helicase （BLM） gene product encodes a DNA helicase that functions in homologous recombination repair to prevent genomic instability. BLM is highly active in binding and unfolding G-quadruplexes （G4）, whi...The Bloom helicase （BLM） gene product encodes a DNA helicase that functions in homologous recombination repair to prevent genomic instability. BLM is highly active in binding and unfolding G-quadruplexes （G4）, which are non- canonical DNA structures formed by Hoogsteen base-pairing in guanine-rich sequences. Here we use single-molecule fluorescence resonance energy transfer （smFRET） to study the molecular mechanism of BLM-catalysed G4 unfolding and show that BLM unfolds G4 in two pathways. Our data enable us to propose a model in which the HRDC domain functions as a regulator of BLM, depending on the position of the HRDC domain of BLM in action： when HRDC binds to the G4 sequence, BLM may hold G4 in the unfolded state; otherwise, it may remain on the unfolded G4 transiently so that G4 can refold immediately.展开更多
A simple, rapid, highly sensitive electrochem-ical sensor for potassium ion （K^＋） based on the confor-mationai change of DNA sequence containing guanine-rich segments is presented. In the presence of K^＋, guanine-...A simple, rapid, highly sensitive electrochem-ical sensor for potassium ion （K^＋） based on the confor-mationai change of DNA sequence containing guanine-rich segments is presented. In the presence of K^＋, guanine-rich DNA sequence folds to G-quadruplex structure, allowing a ferrocene tag to transfer electrons to the electrode. Gold nanoparticles （AuNPs）, which are self-assembled on the surface of a bare gold electrode by using 4-aminothio-phenol as a medium, offer a big surface area to immobilize a large number of aptamers and improve the sensitivity of the sensor. The square-wave voltammetry peak current increases with K^＋ concentration. The plots of peak current against K^＋ concentration and the logarithm of K^＋ con- centration are linear over the range from 0.1 to 1.0 mmol·L^-1 and from 1 to 30 mmol·L^-1, respectively. A lower detection limit of 0.1 mmol·L^-1 K^＋ is obtained for AuNPs-modified sensor, which greatly surpasses that （100 mmol·L^-1） of the sensor without AuNPs modification by three orders of magnitude. Thus, the sensor with AuNPs amplification is expected to open new opportunities for highly sensitive detection of other biomolecules in the future.展开更多
Human telomeric G-quadruplex plays a crucial role in regulating the genome stability. Despite extensive studies on structures and kinetics of monomeric G-quadruplex, the interaction between G-quadruplexes is still in ...Human telomeric G-quadruplex plays a crucial role in regulating the genome stability. Despite extensive studies on structures and kinetics of monomeric G-quadruplex, the interaction between G-quadruplexes is still in debate. In this work,we employ magnetic tweezers to investigate the folding and unfolding kinetics of two contiguous G-quadruplexes in 100-mM K~＋buffer. The interaction between G-quadruplexes and the consequent effect on the kinetics of G-quadruplex are revealed. The linker sequence between G-quadruplexes is further found to play an important role in the interaction between two G-quadruplexes. Our results provide a high-resolution insight into kinetics of multimeric G-quadruplexes and genome stability.展开更多
The detection of biomarkers is of great significance in the diagnosis of numerous diseases,especially cancer.Herein,we developed a sensitive and universal fluorescent aptasensor strategy based on magnetic beads,DNA G-...The detection of biomarkers is of great significance in the diagnosis of numerous diseases,especially cancer.Herein,we developed a sensitive and universal fluorescent aptasensor strategy based on magnetic beads,DNA G-quadruplex,and exonuclease Ⅲ(Exo Ⅲ).In the presence of a target protein,a label-free single strand DNA(ssDNA)hybridized with the aptamer was released as a trigger DNA due to specific recognition between the aptamer and target.Subsequently,ssDNA initiates the ExoⅢ-aided recycling to amplify the fluorescence signal,which was caused by N-methylmesoporphyrin IX(NMM)insertion into the G-quadruplex structure.This proposed strategy combines the excellent specificity between the aptamer and target,high sensitivity of the fluorescence signal by G-quadruplex and ExoⅢ-aided recycling amplification.We selected(50-1200 nmol/L)MUC1,a common tumor biomarker,as the proof-of-concept target to test the specificity of our aptasenso r.Results reveal that the sensor sensitively and selectively detected the target protein with limits of detection(LODs)of 3.68 and 12.83 nmol/L in buffer solution and 10%serum system,respectively.The strategy can be easily applied to other targets by simply substituting corresponding aptamers and has great potential in the diagnosis and monitoring of several diseases.展开更多
The human telomere sequence (TTAGGG)4 folds into an unusual conformation possessing three G-tetrads linked by TTA loops. The first loop is a propeller loop while the second and third loops are transverse loops. Using ...The human telomere sequence (TTAGGG)4 folds into an unusual conformation possessing three G-tetrads linked by TTA loops. The first loop is a propeller loop while the second and third loops are transverse loops. Using Circular Dichroism (CD) spectroscopy, we have investigated the effect of sequence context on the structures and stabilities of intramolecular G-quadruplexes related to the human telomere sequence by considering all permutations of T and A within the loops. The results indicate that changing only one base in any one loop can have a dramatic effect on the conformation of the quadruplex as well as its melting temperature, Tm. Thus, each sequence studied has a unique CD spectrum and Tm. In general, variants with a modified second loop are the most stable while the wild type sequence is the least stable. The observed difference in CD spectra and melting temperature are discussed in terms of base stacking within the loop and stacking of the loop bases with adjacent G-tetrads.展开更多
Mercury(Hg^(2+))has been recognized as a global pollutant with a toxic,mobile,and persistent nature.It adversely affects the ecosystem and human health.Already developed biosensors for Hg^(2+)detection majorly suffer ...Mercury(Hg^(2+))has been recognized as a global pollutant with a toxic,mobile,and persistent nature.It adversely affects the ecosystem and human health.Already developed biosensors for Hg^(2+)detection majorly suffer from poor sensitivity and specificity.Herein,a colorimetric/fluorimetric dual-mode sensing approach is designed for the quantitative detection of Hg^(2+).This novel sensing approach utilizes nanofluorophores,i.e.,fluorescent copper nanoclusters-doped zirconia metal-organic framework(CuNCs@Zr-MOF)nanoconjugate(blue color)and N-methyl mesoporphyrin IX(NMM)(red color)in combination with peroxidase-mimicking G-quadruplex DNAzyme(PMDNAzyme).In the presence of Hg^(2+),dabcyl conjugated complementary DNA with T-T mismatches form the stable duplex with the CuNCs@Zr-MOF@G-quadruplex structure through T-Hg^(2+)-T base pairing.It causes the quenching of fluorescence of CuNCs@Zr-MOF(463 nm)due to the Förster resonance energy transfer(FRET)system.Moreover,the G-quadruplex(G4)structure of the aptamer enhances the fluorescence emission of NMM(610 nm).Besides this,the peroxidase-like activity of G4/hemin DNAzyme offers the colorimetric detection of Hg^(2+).The formation of duplex with PMDNAzyme increases the catalytic activity.This novel biosensing probe quantitatively detected Hg^(2+)using both fluorimetry and colorimetry approaches with a low detection limit of 0.59 and 36.3 nM,respectively.It was also observed that the presence of interfering metal ions in case of real aqueous samples does not affect the performance of this novel biosensing probe.These findings confirm the considerable potential of the proposed biosensing probe to screen the concentration of Hg^(2+)in aquatic products.展开更多
Introduction:A quadruplex digital polymerase chain reaction(dPCR)method was developed for the simultaneous detection of Salmonella spp.,Shigella spp.,Vibrio cholerae,and V.parahaemolyticus in wastewater to enhance pat...Introduction:A quadruplex digital polymerase chain reaction(dPCR)method was developed for the simultaneous detection of Salmonella spp.,Shigella spp.,Vibrio cholerae,and V.parahaemolyticus in wastewater to enhance pathogen identification velocity and efficiency.This study established detection limits for these bacterial pathogens and validated the method using environmental wastewater samples.Methods:Specific primers and probes were designed targeting the invA gene of Salmonella,ipaH gene of Shigella,tlh gene of V.parahaemolyticus,and cholera toxin gene ctxA of V.cholerae.The quadruplex dPCR assay underwent rigorous evaluation for analytical sensitivity and specificity.Detection limits were determined using spiked wastewater samples,and the method’s effectiveness was assessed through preliminary testing of 60 environmental wastewater samples.Results:The quadruplex dPCR assay was optimized at an annealing temperature of 58°C.In spiked wastewater samples,the detection limits were 390 CFU/100 mL for Salmonella,11 CFU/100 mL for Shigella,660 CFU/100 mL for V.cholerae,and 640 CFU/100 mL for V.parahaemolyticus.Analysis of 60 municipal wastewater samples revealed pathogen concentrations ranging from 100.9–14,560 copies/100 mL for Shigella,86.5–7,329 copies/100 mL for Salmonella,and 84.5–865.7 copies/100 mL for V.parahaemolyticus.Conclusions:The developed quadruplex dPCR assay demonstrates robust capability for comprehensive surveillance of intestinal bacterial pathogens in wastewater,offering reliable detection even at low concentrations.展开更多
In the study of the fabrication of DNA-templated silver nanoclusters （DNA-Ag NCs）, how templates affect the fluorescence of the nanoclusters remains undear, and it has been a challenge to understand the correlation ...In the study of the fabrication of DNA-templated silver nanoclusters （DNA-Ag NCs）, how templates affect the fluorescence of the nanoclusters remains undear, and it has been a challenge to understand the correlation between the properties of the DNA template and the Ag NCs. In this respect, based on the rational design of a series of structurally defined intramolecular G-quadruplexes, we prepared G-quadruplex-templated Ag NCs with a defined G-tetrad-to-silver ratio of 1：2. We evaluated the effect of G-quadruplex topology and loop sequences on the fluorescence of DNA-Ag NCs using circular dichroism, and extinction and emission spectroscopy. G-quadruplex templates with an anti-parallel topology were found to produce Ag NCs with stronger fluorescence compared with parallel and hybrid configurations. Loop bases adjacent to G-tetrads have a more significant impact on the fluorescence of Ag NCs compared with those in the middle of the loop, with adenine largely exhibiting an enhancement effect and thymine being detrimental. Generally, G-quadruplexes having an anti-parallel topology with adenine in the loop adjacent to the G-tetrad would be good templates for producing highly fluorescent Ag NCs. This is the first study to focus on the correlation between G-quadruplex topology/sequence and the optical properties of Ag NCs. We hope that the results of this study will facilitate a more in-depth understanding of correlation between G-quadruplex templates and Ag NCs, and help to understand and utilize their unique attributes.展开更多
The binding properties between meso-tetrakis(4-(N-methylpyridiumyl))porphyrin (TMPyP4) and the parallel DNA G-quadruplex (G4) of telomeric repeated sequence 5′-TTAGGG-3′ have been characterized by means of circular ...The binding properties between meso-tetrakis(4-(N-methylpyridiumyl))porphyrin (TMPyP4) and the parallel DNA G-quadruplex (G4) of telomeric repeated sequence 5′-TTAGGG-3′ have been characterized by means of circular dichroism,steady-state absorption,steady-state fluorescence and picosecond time-resolved fluorescence spectroscopies. The binding constant and the saturated binding number were determined as 1.29×106 (mol/L)-1 and 3,respectively,according to steady-state absorption spec-troscopy. Based on the findings by the use of time-resolved fluorescence spectroscopic technique,it is deduced that TMPyP4 binds to a DNA G-quadruplex with both the thread-intercalating and end-stacking modes and at the saturated binding state,one TMPyP4 molecule intercalates into the intervals of G-tetrads while the other two stack to the ends of the DNA G-quadruplex.展开更多
文摘The influence of the G‐quartet structural integrity on the catalytic activity of the G‐quadruplex(G4)was investigated by comparing the G4‐DNAzyme performances of a series of G4s with a G‐vacancy site and a G‐triplex(G‐tri).The results presented herein not only confirm that the structural integrity of the 3'‐end G‐quartet is necessary for G4s to be catalytically competent but also show how to remediate G‐vacancy‐mediated catalytic activity losses via the addition of guanine surrogates in an approach referred to as G‐vacancy complementation strategy that is applicable to parallel G4s only.Furthermore,this study demonstrates that the terminal G‐quartet could act as a proximal coordinating group and cooperate with the flanking nucleotide to activate the hemin cofactor.
基金supported by the grants from the National Natural Science Foundation of China (31720103912 and 31801704)the ’Taishan Scholar’ Construction Project, China (TS201712023)。
文摘Tomato brown rugose fruit virus(ToBRFV) is a novel tobamovirus firstly reported in 2015 and poses a severe threat to the tomato industry. So far, it has spread to 10 countries in America, Asia, and Europe. In 2019, ToBRFV was identified in Shandong Province(ToBRFV-SD), China. In this study, it was shown that ToBRFV-SD induced mild to severe mosaic and blistering on leaves, necrosis on sepals and pedicles, and deformation, yellow spots, and brown rugose necrotic lesions on fruits. ToBRFV-SD induced distinct symptoms on plants of tomato, Capsicum annumm, and Nicotiana benthamiana, and caused latent infection on plants of Solanum tuberosum, Solanum melongena, and N. tabacum cv. Zhongyan 102. All the 50 tomato cultivars tested were highly sensitive to ToBRFV-SD. The complete genomic sequence of ToBRFV-SD shared the highest nucleotide and amino acid identities with isolate IL from Israel. In the phylogenetic tree constructed with the complete genomic sequence, all the ToBRFV isolates were clustered together and formed a sister branch with tobacco mosaic virus(TMV). Furthermore, a quadruplex RT-PCR system was developed that could differentiate ToBRFV from other economically important viruses affecting tomatoes, such as TMV, tomato mosaic virus, and tomato spotted wilt virus. The findings of this study enhance our understanding of the biological and molecular characteristics of ToBRFV and provide an efficient and effective detection method for multiple infections, which is helpful in the management of ToBRFV.
基金supported by USDA-ARS and grants from the National Science Foundation(PGRP IOS-1025954 to HWB,PGRP IOS1116561 to KEK and coworkers)the U.S.Department of Agriculture(NRI-Plant Biochemistry 07-03580 to KEK and coworkers)The Florida State University(CRC Planning Grant to HWB,OMNI award No.0000025471)
文摘The G-quadruplex （G4） elements comprise a class of nucleic acid structures formed by stacking of guanine base quartets in a quadruple helix. This （34 DNA can form within or across single-stranded DNA molecules and is mutually exclusive with duplex B-form DNA. The reversibility and structural diversity of G4s make them highly versatile genetic structures, as demonstrated by their roles in various functions including telomere metabolism, genome maintenance, immunoglobulin gene diversification, transcription, and translation. Sequence motifs capable of forming G4 DNA are typically located in telomere repeat DNA and other non-telomeric genomic loci. To investigate their potential roles in a large-genome model plant species, we computationaily identified 149,988 non-telomeric G4 motifs in maize （Zea mays L., B73 AGPv2）, 29% of which were in non-repetitive genomic regions. G4 motif hotspots exhibited non-random enrichment in genes at two locations on the antisense strand, one in the 5~ UTR and the other at the 5~ end of the first intron. Several genic G4 motifs were shown to adopt sequence-specific and potassium-dependent G4 DNA structures in vitro. The G4 motifs were prevalent in key regulatory genes associated with hypoxia （group VII ERFs）, oxidative stress （D J-1/GATasel）, and energy status （AMPK/ SnRK） pathways. They also showed statistical enrichment for genes in metabolic pathways that function in glycolysis, sugar degradation, inositol metabolism, and base excision repair. Collectively, the maize G4 motifs may represent conditional regulatory elements that can aid in energy status gene responses. Such a network of elements could provide a mechanistic basis for linking energy status signals to gene regulation in maize, a model genetic system and major world crop species for feed, food, and fuel.
基金supported by National Natural Science Foundation of China (Nos. 21205142, 31370104)The Research Innovation Program for Graduates of Central South University (No. 2016zzts580)
文摘In this assay, a label-free fluorescent sensing platform based on triple-helix molecular switch（THMS） and G-quadruplex was developed for the detection of tetracycline. We demonstrated this approach by using THMS, which consists of a central section with a shortened 8-mer aptamer sequence with high affinity to tetracycline and flanked by two arm segments. G-rich oligonucleotide can specifically bind to thioflavin T（Th T） as a signal transduction probe（STP）. In the absence of tetracycline, THMS remains stable, the fluorescence of background is low. By the addition of target tetracycline, the aptamer-target binding results in the formation of a structured aptamer-target complex, which disassembles the THMS and releases the STP. The free STP self-assembles into G-quadruplex and specifically binds to Th T which generates a obvious fluorescence enhancement. Using the triple-helix molecular switch, the developed aptamer-based fluorescent sensing platform showed a linear relationship with the concentration of tetracycline ranging from 0.2 to 20.0 nmol/L. The detection limit of tetracycline was determined to be970.0 pmol/L. The assay avoids complicated modifications or chemical labeling, making it simple and cost-effective. So, it is expected that this aptamer-based fluorescent assay could be extensively applied in the field of food safety inspection.
基金supported by the National Natural Science Foundation of China(Nos90913004 and 30821001)the Natural Science Foundation of Beijing(No7082101)+1 种基金Program for New Century Excellent Talents in UniversityBeijing Talents Foundation Beijing National Laboratory for Molecular Sciences
文摘The G-quadruplexes formed from G-rich strands in the telomere and oncogene-promoter regions are regarded as new promising targets in the cancer therapy.A G-quadruplex in the downstream flanking region of the signal transducer and activator of transcription 3（STAT3） gene was explored.Its folding patterns were proposed to be 3：2：2 and 3：3：1 loop isomers by the mutation analysis by CD spectroscopy.The structures were constructed and refined by molecular modeling method.
基金the National Natural Science Foundation of China(No.20472009)the Research Fund for the Doctoral Program of Higher Education.
文摘In this research, an unusually dimeric G-quadruplex of d（GGGTGGGTGGGTGGGT） （SI）, the potent nanomolar HIV-1 integrase inhibitor, was detected by nuclear magnetic resonance （NMR）. This result has been confirmed by electrospray ionization mass spectrometry （ESI-MS） and circular dichroism （CD）.
基金supported by Major National Basic Research Projects (973,No.2013CB733701)National Natural Science Foundation of China (Nos.81072576,91027033,21302188,21205121,21305145 and 31200576)Chinese Academy of Sciences (No.KJCX2-EW-N06-01)
文摘G-quadruplexes （G4s） play important roles in biological systems, such as telomere maintenance, replication, and transcription. Based on the DNA sequence, loop geometry, and the local environments, G4s can be classified into different conformations. It is important to detect different types of G4s to monitor the diseases related with G4s. Most ligands bind to G4s based on end-stacking modes, while rare ligands bind to G4s through groove binding modes. We have found that a cyanine dye DMSB interacts with parallel G4 by end-stacking and groove simultaneous binding mode. In this article, we found that DMSB could simply discriminate parallel G4s from other DNA motifs by using UV-vis spectrum. These results give some clues to develop high specificity G4 probes.
基金financially supported by the National Natural Science Foundation of China(No. 21475091)the Science andTechnology Department of Sichuan Province(No. 2015GZ0301)
文摘G-quadruplex ligands have been accepted as potential therapeutic agents for anticancer treatment. Thioflavin T （ThT）, a highly selective G-quadruplex ligand, can bind G-quadruplex with a fluorescent light-up signal change and high specificity against DNA duplex. However, there are still different opinions that ThT induces/stabilizes G-quadruplex foldings/topologies in human telomere sequence. Here, a sensitive single-molecule nanopore technology was utilized to analyze the interactions between human telomeric DNA （Tel DNA） and ThT. Both translocation time and current blockade were measured to investigate the translocation behaviors. Furthermore, the effects of metal ion （K~ and Na~） and pH on the translocation behaviors were studied. Based on the single-molecule level analysis, there are some conclusions： （1） In the electrolyte solution containing 50 mmol/L I（Cl and 450 mmol/L NaCl, ThT can bind strongly with Tel DNA but nearly does not change the G-quadruplex form; （2） in the presence of high concentration K~, the ThT binding induces the structural change of hybrid G-quadruplex into antiparallel topology with an enhanced structural stability; （3） In either alkaline or acidic buffer, G-quadruplex form will change in certain degree. All above conclusions are helpful to deeply understand the interaction behaviors between Tel DNA and ThT. This nanopore platform, investigating G-quadruplex ligands at the single-molecule level, has great potential for the design of new drugs and sensors.
文摘The DNA G quadruplex formed by the human telomeric sequence is a potential target for novel anticancer drugs. We have investigated an intramolecular DNA G quadruplex using single molecule fluorescence resonance energy transfer and shown that individual folded quadruplexes can be identified. The mean proximity ratio measured at the single molecule level was consistent with ensemble measurement
基金supported by the National Natural Science Foundation of China(Grant Nos.11674382,11574381,and 11574382)the Key Research Program of Frontier Sciences,Chinese Academy of Sciences(Grant No.QYZDJ-SSW-SYS014)
文摘The Bloom helicase （BLM） gene product encodes a DNA helicase that functions in homologous recombination repair to prevent genomic instability. BLM is highly active in binding and unfolding G-quadruplexes （G4）, which are non- canonical DNA structures formed by Hoogsteen base-pairing in guanine-rich sequences. Here we use single-molecule fluorescence resonance energy transfer （smFRET） to study the molecular mechanism of BLM-catalysed G4 unfolding and show that BLM unfolds G4 in two pathways. Our data enable us to propose a model in which the HRDC domain functions as a regulator of BLM, depending on the position of the HRDC domain of BLM in action： when HRDC binds to the G4 sequence, BLM may hold G4 in the unfolded state; otherwise, it may remain on the unfolded G4 transiently so that G4 can refold immediately.
基金financially supported by the National Natural Science Foundation of China(No.20903008)
文摘A simple, rapid, highly sensitive electrochem-ical sensor for potassium ion （K^＋） based on the confor-mationai change of DNA sequence containing guanine-rich segments is presented. In the presence of K^＋, guanine-rich DNA sequence folds to G-quadruplex structure, allowing a ferrocene tag to transfer electrons to the electrode. Gold nanoparticles （AuNPs）, which are self-assembled on the surface of a bare gold electrode by using 4-aminothio-phenol as a medium, offer a big surface area to immobilize a large number of aptamers and improve the sensitivity of the sensor. The square-wave voltammetry peak current increases with K^＋ concentration. The plots of peak current against K^＋ concentration and the logarithm of K^＋ con- centration are linear over the range from 0.1 to 1.0 mmol·L^-1 and from 1 to 30 mmol·L^-1, respectively. A lower detection limit of 0.1 mmol·L^-1 K^＋ is obtained for AuNPs-modified sensor, which greatly surpasses that （100 mmol·L^-1） of the sensor without AuNPs modification by three orders of magnitude. Thus, the sensor with AuNPs amplification is expected to open new opportunities for highly sensitive detection of other biomolecules in the future.
基金supported by the National Natural Science Foundation of China(Grant Nos.11474346 and 11774407)the Key Research Program of Frontier Sciences,Chinese Academy of Sciences(Grant No.QYZDB-SSW-SLH045)the National Key Research and Development Program,China(Grant No.2016YFA0301500)
文摘Human telomeric G-quadruplex plays a crucial role in regulating the genome stability. Despite extensive studies on structures and kinetics of monomeric G-quadruplex, the interaction between G-quadruplexes is still in debate. In this work,we employ magnetic tweezers to investigate the folding and unfolding kinetics of two contiguous G-quadruplexes in 100-mM K~＋buffer. The interaction between G-quadruplexes and the consequent effect on the kinetics of G-quadruplex are revealed. The linker sequence between G-quadruplexes is further found to play an important role in the interaction between two G-quadruplexes. Our results provide a high-resolution insight into kinetics of multimeric G-quadruplexes and genome stability.
基金supported by grants from the National Natural Science Foundation of China (No.21472060)Shenzhen Municipal government (Nos.JCYJ20160301153959476 and JCYJ20160324163734374)Shenzhen Reform Commission (Disciplinary Development Program for Chemical Biology)
文摘The detection of biomarkers is of great significance in the diagnosis of numerous diseases,especially cancer.Herein,we developed a sensitive and universal fluorescent aptasensor strategy based on magnetic beads,DNA G-quadruplex,and exonuclease Ⅲ(Exo Ⅲ).In the presence of a target protein,a label-free single strand DNA(ssDNA)hybridized with the aptamer was released as a trigger DNA due to specific recognition between the aptamer and target.Subsequently,ssDNA initiates the ExoⅢ-aided recycling to amplify the fluorescence signal,which was caused by N-methylmesoporphyrin IX(NMM)insertion into the G-quadruplex structure.This proposed strategy combines the excellent specificity between the aptamer and target,high sensitivity of the fluorescence signal by G-quadruplex and ExoⅢ-aided recycling amplification.We selected(50-1200 nmol/L)MUC1,a common tumor biomarker,as the proof-of-concept target to test the specificity of our aptasenso r.Results reveal that the sensor sensitively and selectively detected the target protein with limits of detection(LODs)of 3.68 and 12.83 nmol/L in buffer solution and 10%serum system,respectively.The strategy can be easily applied to other targets by simply substituting corresponding aptamers and has great potential in the diagnosis and monitoring of several diseases.
文摘The human telomere sequence (TTAGGG)4 folds into an unusual conformation possessing three G-tetrads linked by TTA loops. The first loop is a propeller loop while the second and third loops are transverse loops. Using Circular Dichroism (CD) spectroscopy, we have investigated the effect of sequence context on the structures and stabilities of intramolecular G-quadruplexes related to the human telomere sequence by considering all permutations of T and A within the loops. The results indicate that changing only one base in any one loop can have a dramatic effect on the conformation of the quadruplex as well as its melting temperature, Tm. Thus, each sequence studied has a unique CD spectrum and Tm. In general, variants with a modified second loop are the most stable while the wild type sequence is the least stable. The observed difference in CD spectra and melting temperature are discussed in terms of base stacking within the loop and stacking of the loop bases with adjacent G-tetrads.
基金Funding:S.K.thanks the Department of Biotechnology(DBT),Government of India,for research grant(award BT/PR18868/BCE/8/1370/2016 dated 2018 January 31)M.N.is grateful to CSIR for the SRA fellowship(no.B-12857,dated 2021 October 21).
文摘Mercury(Hg^(2+))has been recognized as a global pollutant with a toxic,mobile,and persistent nature.It adversely affects the ecosystem and human health.Already developed biosensors for Hg^(2+)detection majorly suffer from poor sensitivity and specificity.Herein,a colorimetric/fluorimetric dual-mode sensing approach is designed for the quantitative detection of Hg^(2+).This novel sensing approach utilizes nanofluorophores,i.e.,fluorescent copper nanoclusters-doped zirconia metal-organic framework(CuNCs@Zr-MOF)nanoconjugate(blue color)and N-methyl mesoporphyrin IX(NMM)(red color)in combination with peroxidase-mimicking G-quadruplex DNAzyme(PMDNAzyme).In the presence of Hg^(2+),dabcyl conjugated complementary DNA with T-T mismatches form the stable duplex with the CuNCs@Zr-MOF@G-quadruplex structure through T-Hg^(2+)-T base pairing.It causes the quenching of fluorescence of CuNCs@Zr-MOF(463 nm)due to the Förster resonance energy transfer(FRET)system.Moreover,the G-quadruplex(G4)structure of the aptamer enhances the fluorescence emission of NMM(610 nm).Besides this,the peroxidase-like activity of G4/hemin DNAzyme offers the colorimetric detection of Hg^(2+).The formation of duplex with PMDNAzyme increases the catalytic activity.This novel biosensing probe quantitatively detected Hg^(2+)using both fluorimetry and colorimetry approaches with a low detection limit of 0.59 and 36.3 nM,respectively.It was also observed that the presence of interfering metal ions in case of real aqueous samples does not affect the performance of this novel biosensing probe.These findings confirm the considerable potential of the proposed biosensing probe to screen the concentration of Hg^(2+)in aquatic products.
基金Supported by the National Key Research and Development Program(2022YFC3201704-01)from the Ministry of Science and Technology of the People’s Republic of Chinathe Key Project of the Capital’s Funds for Health Improvement and Research,China(2022-1G-4231).
文摘Introduction:A quadruplex digital polymerase chain reaction(dPCR)method was developed for the simultaneous detection of Salmonella spp.,Shigella spp.,Vibrio cholerae,and V.parahaemolyticus in wastewater to enhance pathogen identification velocity and efficiency.This study established detection limits for these bacterial pathogens and validated the method using environmental wastewater samples.Methods:Specific primers and probes were designed targeting the invA gene of Salmonella,ipaH gene of Shigella,tlh gene of V.parahaemolyticus,and cholera toxin gene ctxA of V.cholerae.The quadruplex dPCR assay underwent rigorous evaluation for analytical sensitivity and specificity.Detection limits were determined using spiked wastewater samples,and the method’s effectiveness was assessed through preliminary testing of 60 environmental wastewater samples.Results:The quadruplex dPCR assay was optimized at an annealing temperature of 58°C.In spiked wastewater samples,the detection limits were 390 CFU/100 mL for Salmonella,11 CFU/100 mL for Shigella,660 CFU/100 mL for V.cholerae,and 640 CFU/100 mL for V.parahaemolyticus.Analysis of 60 municipal wastewater samples revealed pathogen concentrations ranging from 100.9–14,560 copies/100 mL for Shigella,86.5–7,329 copies/100 mL for Salmonella,and 84.5–865.7 copies/100 mL for V.parahaemolyticus.Conclusions:The developed quadruplex dPCR assay demonstrates robust capability for comprehensive surveillance of intestinal bacterial pathogens in wastewater,offering reliable detection even at low concentrations.
基金This work was supported by the National Natural Science Foundation of China （Nos. 21535006, 21475004, and 21275011）.
文摘In the study of the fabrication of DNA-templated silver nanoclusters （DNA-Ag NCs）, how templates affect the fluorescence of the nanoclusters remains undear, and it has been a challenge to understand the correlation between the properties of the DNA template and the Ag NCs. In this respect, based on the rational design of a series of structurally defined intramolecular G-quadruplexes, we prepared G-quadruplex-templated Ag NCs with a defined G-tetrad-to-silver ratio of 1：2. We evaluated the effect of G-quadruplex topology and loop sequences on the fluorescence of DNA-Ag NCs using circular dichroism, and extinction and emission spectroscopy. G-quadruplex templates with an anti-parallel topology were found to produce Ag NCs with stronger fluorescence compared with parallel and hybrid configurations. Loop bases adjacent to G-tetrads have a more significant impact on the fluorescence of Ag NCs compared with those in the middle of the loop, with adenine largely exhibiting an enhancement effect and thymine being detrimental. Generally, G-quadruplexes having an anti-parallel topology with adenine in the loop adjacent to the G-tetrad would be good templates for producing highly fluorescent Ag NCs. This is the first study to focus on the correlation between G-quadruplex topology/sequence and the optical properties of Ag NCs. We hope that the results of this study will facilitate a more in-depth understanding of correlation between G-quadruplex templates and Ag NCs, and help to understand and utilize their unique attributes.
基金the National Natural Science Foundation of China (Grant Nos. 20442004, 10576002 and 20703067)
文摘The binding properties between meso-tetrakis(4-(N-methylpyridiumyl))porphyrin (TMPyP4) and the parallel DNA G-quadruplex (G4) of telomeric repeated sequence 5′-TTAGGG-3′ have been characterized by means of circular dichroism,steady-state absorption,steady-state fluorescence and picosecond time-resolved fluorescence spectroscopies. The binding constant and the saturated binding number were determined as 1.29×106 (mol/L)-1 and 3,respectively,according to steady-state absorption spec-troscopy. Based on the findings by the use of time-resolved fluorescence spectroscopic technique,it is deduced that TMPyP4 binds to a DNA G-quadruplex with both the thread-intercalating and end-stacking modes and at the saturated binding state,one TMPyP4 molecule intercalates into the intervals of G-tetrads while the other two stack to the ends of the DNA G-quadruplex.
