DNAzyme-based gene therapy faces some challenges including cell penetration,activity limitation,and co-delivery functions.Self-assembled DNA nanomedicine has attracted widespread attention due to its many advantages.I...DNAzyme-based gene therapy faces some challenges including cell penetration,activity limitation,and co-delivery functions.Self-assembled DNA nanomedicine has attracted widespread attention due to its many advantages.It is urgent to develop a universal DNA degradation strategy for precise programmable drug release.Herein,we reported a self-catabolic DNAzyme nanospheres(SCNS),which could simultaneously achieve cell penetration,activity enhancement,and co-delivery functions.The SCNS were assembled through Y-DNA stepwise hybridization with each other,which were then loaded with aptamer(Apt),doxorubicin(Dox),and zinc oxide nanoparticles(ZnO NPs).The acid-triggered dissociation of ZnO NPs leads to the generation of Zn^(2+)ions cofactors for immediately self-catabolic DNAzyme nanospheres.After the disassembly of the SCNS,three types of anticancer treatments would be activated,which include Zn^(2+)involved reactive oxygen species(ROS),Dox-induced chemotherapy,and DNAzyme-based gene therapy.The experimental results show that the nanoplatform(Apt-SCNS-Dox-ZnO)has a good tumor-killing effect and minimal side effects.As a smart self-driven drug delivery nanoplatform,it is anticipated to displace extraordinary potential in biomedicine and bioengineering.展开更多
Detection of deoxyribozyme (DNAzyme) cleavage process usually needs complex and time-consuming radial labeling, gel electrophoresis and autoradiography. This paper reported an approach to detect DNAzyme cleavage pro...Detection of deoxyribozyme (DNAzyme) cleavage process usually needs complex and time-consuming radial labeling, gel electrophoresis and autoradiography. This paper reported an approach to detect DNAzyme cleavage process in real time using a fluorescence probe. The probe was employed as DNAzyme substrate to convert directly the cleavage information into fluorescence signal in real time. Compared with traditional approach, this non-isotope method not only brought a convenient means to monitor the DNAzyme cleavage reaction, but also offered abundant dynamic data for choosing potential gene therapeutic agents. It provides a new tool for DNAzyme research, as well as a new insight into research on human disease diagnosis. Based on this method, 8- 17deoxyribozyme (8-17DNAzyme) against hepatitis C virus RNA (HCV-RNA) was designed and the cleavage process was studied in real time. ?2009 Ke Min Wang. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All fights reserved.展开更多
Studying the expression level of mRNA in living cells will offer tremendous opportunities for ad-vancement in cell biology research,disease diagnostics,and drug discovery.In this paper,a molecular beacon(MB)specific f...Studying the expression level of mRNA in living cells will offer tremendous opportunities for ad-vancement in cell biology research,disease diagnostics,and drug discovery.In this paper,a molecular beacon(MB)specific for the important tumor suppressor gene p21 has been designed and synthesized.The fluorescence signal was detected in real-time after the MB entered the cytoplasm of nasopharyn-geal carcinoma cells.After injecting the p21MB into nasopharyngeal carcinoma cell and p33-trans-fected nasopharyngeal carcinoma cell,the consistent increase of fluorescent signal intensity was de-tected in both cell lines,and maximum fluorescence intensity achieved in about 15 min.In about 4 min following microinjection,the fluorescence increasing rate was significantly different between these two cell lines,which indicate the different p21 mRNA expression levels.The results obtained in the real-time detection were also validated by RT-PCR.Analysis of the initial fluorescence increasing rate can effi-ciently reduce the side effect of enzyme and improve the accuracy in living cell mRNA detection.展开更多
In this study, GFP mRNA in COS-7 cell and GFP-transfected COS-7 cell was detected in real time using phosphorothioate-modified molecular beacon based on living cell imaging method. Results showed that phosphorothioate...In this study, GFP mRNA in COS-7 cell and GFP-transfected COS-7 cell was detected in real time using phosphorothioate-modified molecular beacon based on living cell imaging method. Results showed that phosphorothioate-modified molecular beacon still kept the advantages of molecular beacon, such as, excellent selectivity, high sensitivity, and no separation detection. In addition, this modification could significantly increase the nuclease resistance of molecular beacon. Phosphorothioate-modified molecular beacon can efficiently reduce the false positive signal and improve the accuracy of living cell mRNA detection.展开更多
Combining the inhibited aptazyme and molecular beacon (MB), we developed a versatile sensing strategy for amplified detection of adenosine. In this strategy, the adenosine aptamer links to the 8-17 DNAzyme to form a...Combining the inhibited aptazyme and molecular beacon (MB), we developed a versatile sensing strategy for amplified detection of adenosine. In this strategy, the adenosine aptamer links to the 8-17 DNAzyme to form an aptazyme. A short sequence, denoted as inhibitor, is designed to form a duplex spanning the aptamer-DNAzyme junction, which blocks the catalytic function of the DNAzyme. Only in the presence of target adenosine, the aptamer binds to adenosine, thus the inhibitor dissociates from the aptamer portion of the aptazyme and can no longer form the stable duplex required to inhibit the catalytic activity of the aptazyme. The released DNAzyme domain will hybridize to the MB and catalyze the cleavage in the presence of Zn2+, making the fluorophore separate from the quencher and resulting in fluorescence signal. The results showed that the detection method has a dynamic range from 10 nmol/L to 1nmol/L, with a detection limit of 10 nmol/L.展开更多
A one-step and facile strategy toward the construction of multicolor polymers via supramolecular selfassembly was proposed.Multicolor polymers were simply prepared by the self-assembly of adamantane-labeled fluorescei...A one-step and facile strategy toward the construction of multicolor polymers via supramolecular selfassembly was proposed.Multicolor polymers were simply prepared by the self-assembly of adamantane-labeled fluorescein,adamantane-labeled rhodamine B and β-cyclodextrin polymers via host-guest interaction between β-cyclodextrin and adamantane.Multicolor polymers showed several interesting properties:multiple emission signatures by a single wavelength excitation,easy tunability,intense fluorescence,high photostablility.In addition,the self-assembly approach implied a facile and flexible strategy for constructing functionalized materials,such as multicolor materials for biological labeling and imaging,and sensing materials for the detection of physiological parameters.展开更多
Implanting artificial organelles in living cells is capable of correcting cellular dysfunctionalities for cell repair and biomedical applications. In this work, phase-separated bienzyme-loaded coacervate microdroplets...Implanting artificial organelles in living cells is capable of correcting cellular dysfunctionalities for cell repair and biomedical applications. In this work, phase-separated bienzyme-loaded coacervate microdroplets are established as a model of artificial membraneless organelles in endothelial dysfunctional cells for the cascade enzymatic production of nitric oxide(NO) with a purpose of correcting cellular NO deficiency. We prepared the coacervate microdroplets via liquid-liquid phase separation of oppositely charged polyelectrolytes, in which glucose oxidase/horseradish peroxidase-mediated cascade reaction was compartmented. After the coacervate microdroplets were implanted in NO-deficient dysfunctional cells, the compartments maintained a phase-separated liquid droplet structure, which facilitated a significant enhancement of NO production in the dysfunctional cells. The recovery of NO production was further exploited to inhibit clot formation in blood plasma located in the cell suspension. This demonstrated a proof-of-concept design of artificial organelles in dysfunctional cells for cell repair and anticoagulation-related medical applications. Our results demonstrate an approach for the construction of coacervate droplets through phase separation for the generation of artificial membraneless organelles, which can be designed to provide an array of functionalities in living organisms that have the potential to be used in the field of cell engineering and medical therapy.展开更多
Accurate detection and imaging of adenosine triphosphate(ATP)expression levels in living cells is of great value for understanding cell metabolism,physiological activities,and pathologic mechanisms.Here,we developed a...Accurate detection and imaging of adenosine triphosphate(ATP)expression levels in living cells is of great value for understanding cell metabolism,physiological activities,and pathologic mechanisms.Here,we developed a DNA tetrahedron-based split aptamer probe(TD probe)for ratiometric fluorescence imaging of ATP in living cells.The TD probe is constructed by hybridizing two split ATP aptamer probes(Apt-a and Apt-b)to a DNA tetrahedron assembled by four DNA oligonucleotides(T1,T2,T3 and T4).In the presence of ATP,the TD probe will alter its structure from the open to closed state,thus bringing the separated donor and acceptor fluorophores into close proximity for high fluorescence resonance energy transfer(FRET)signals.The TD probe exhibits low cytotoxicity,efficient cell internalization and good biological stability.Moreover,based on the FRET“off”to“on”signal output mode,the TD probe can effectively avoid false-positive signals from complex biological matrices,which is significant for long-term reliable imaging in living cells.In addition,by changing the split aptamers attached to DNA tetrahedron,the proposed strategy may be extended for detecting various intracellular targets.Collectively,this strategy provides a valuable sensing platform for biomarkers analysis in living cells,thus having great potential for early clinical diagnosis and therapeutic evaluation.展开更多
Accurate and sensitive detection of caner cells is of significant importance for early diagnosis and treat-ment of cancer.Here,we developed an extracellular ATP-dctivated hybridization chain reaction(HCR)amplification...Accurate and sensitive detection of caner cells is of significant importance for early diagnosis and treat-ment of cancer.Here,we developed an extracellular ATP-dctivated hybridization chain reaction(HCR)amplification strategy to meet this purpose.This strategy relies on three DNA probes,Apt-trigger,H1-AТP aptamer duplex and hairpin H2.The Apt-trigger probe consists of two com sequence for specific recognition of the target cells.and a trigger sequence for the HCR assembly.Theроnents:an aptamer duplex structure of H1-ATP aptamer causes the tochold in hairpin H1 to be hidden,preventing the strand-ent displacement reaction between haipin H1 and Apt-trigger.Upon activation with ATP the ATP aptamer will blnd to ATP to dissoci iate from hairpin H1,thus leading to an Apt-trigger-induced strand-displacement reaction and subsequent HCR with hairpin H2 on the target cell surface.Benefiting from aptamer recogni-tion and ATP-activated HCR amplification,this strategy can not only perform sensitive quantitative anal-ysis with a detection limit of 25 cells in 200 ul.of binding buffer,but also show desirable specificity and accuracy for identifylng target cells from control cells and mixed cell samples,Imporantly,this method retains stable and good perfor mance for target cell detection in 10%fetal bovine ser rum,den onstrating great potential for clinical diagnosis in complex biological matrices.Furthermore,this strategy can be adapted to detect various types of cancer cells by changing the corresponding aptamer sequence.展开更多
In recent years, specific detection of proteins is one of the hot issues about aptamers in proteomics. Here we reported a simple, sensitive and specific proximity-dependent protein assay with dual DNA aptamers. Thromb...In recent years, specific detection of proteins is one of the hot issues about aptamers in proteomics. Here we reported a simple, sensitive and specific proximity-dependent protein assay with dual DNA aptamers. Thrombin was used as the model protein, and two aptamer probes with complementary se- quence at 3′-end were designed for the two distinct epitopes of the protein. Association of the two ap- tamers with thrombin resulted in stable hybrids due to the proximity of 3′-end, then polymerase reac- tion was induced. The amount of obtained dsDNA was indicated using the fluorescence dye Sybr Green I. The results showed that the initial velocity of polymerase reaction had a positive correlation with concentration of thrombin. The advantages of this dual-aptamer-based approach included simple and flexible design of aptamer probes, high selectivity and high sensitivity. The detection limit was 6.9 pmol/L.展开更多
In clinical cancer research,it is quite promising to develop multimodal synergistic therapeutic strategies.Photodynamic and photothermal synergistic therapy is a very desirable multimodal therapy strategy.Herein,we re...In clinical cancer research,it is quite promising to develop multimodal synergistic therapeutic strategies.Photodynamic and photothermal synergistic therapy is a very desirable multimodal therapy strategy.Herein,we report a facile and simple method to construct a nanotherapeutic agent for photodynamic and photothermal therapy.This nanotherapeutic agent(ZnO@Ce6-PDA)is composed of a ZnO nanoparticle core,an interlayer of photosensitizer chlorin e6(Ce6)and an outer layer of polydopamine(PDA).Due to the existence of Ce6,the ZnO@Ce6-PDA can efficiently generate singlet oxygen(1O2)under 660 nm laser irradiation.Moreover,the ZnO@Ce6-PDA can serve as a photothermal agent,because of the excellent photothermal conversion efficiency of the PDA coating layer in the presence of 780 nm laser.Experiment results demonstrated that the designed nanotherapeutic agent had outstanding phototoxicity upon the combination of laser irradiation at 660 and 780 nm.Thus,our work proves that the ZnO@Ce6-PDA is a promising photodynamic/photothermal dual-modal nanotherapeutic agent for enhanced cancer therapy.展开更多
This work develops a fluorescence approach for sensitive detection of DNA methyltransferase activity based on endonuclease and rolling circle amplification (RCA) technique. In the presence of DNA adenine methylation...This work develops a fluorescence approach for sensitive detection of DNA methyltransferase activity based on endonuclease and rolling circle amplification (RCA) technique. In the presence of DNA adenine methylation (Dam) MTase, the methylation-responsive sequence of hairpin probe is methylated and cleaved by the methylation-sensitive restriction endonuclease Dpn 1. The products cleaved by restriction endonuclease Dpn I then function as a signal primer to initiate RCA reaction by hybridizing with the circular DNA template. Each RCA product containing thousands of repeated sequences might hybridize with a large number of molecular beacons (detection probes), resulting in an enhanced fluorescence signal. In the absence of Dam MTase, neither methylation/cleavage nor RCA reaction can be initiated and no fluorescence signal is observed. The proposed method exhibits a dynamic range from 0.5 U/mL to 30 U/mL and a detection limit of 0.18 U/mL. This method can be used for the screening of antimicrobial drugs and has a great potential to be further applied in early clinical diagnosis.展开更多
Biomimetic nanodevice is an important branch to expand the potential applications of artificial nanochannels. Here, we constructed nanochannels for cascade response of borate and cis-hydroxyl compounds by modifying th...Biomimetic nanodevice is an important branch to expand the potential applications of artificial nanochannels. Here, we constructed nanochannels for cascade response of borate and cis-hydroxyl compounds by modifying the nanochannels of track-etched polycarbonate (TEPC) membranes with polyvinyl alcohol (PVA). Firstly, borate bound to PVA and increased the negative charge density on the surface of the nanochannels, which obstructed the transport of 1,5-naphthalene disulfonate (NDS2-). Subsequently, the addition of cis-hydroxyl compounds induced leaving of borate due to the stronger binding affinities between borate and cis-hydroxyl compounds, which reduced the negative charge density on nanochannels and then enhanced the transport of NDS2-. The cascade response of the nanochannels also accord with the properties of IMP (implication) logic gate. In addition, such nanochannels showed good reproducibility and reversibility. Therefore, this cascade response model based on nanochannels has the potential to be used as switches in area of actuators and biosensors, and is also expected to be used to understand the interaction of substances in nanoscale and simulate the physiological functions of boron.展开更多
Herein, we described a ratiometric strategy based on "chameleon" DNA-silver nanoclusters( DNA-AgNCs) fluorescent binary probes. The strategy was applied to detect high-risk human papillomavirus( HPV) DNA seq...Herein, we described a ratiometric strategy based on "chameleon" DNA-silver nanoclusters( DNA-AgNCs) fluorescent binary probes. The strategy was applied to detect high-risk human papillomavirus( HPV) DNA sequences, HPV-16. First, DNA-AgNCs were synthesized by a simple reduction method. The obtained nanoprobes showed typical yellow and red fluorescence of AgNCs. Upon the addition of HPV-16 DNA, the yellow fluorescence of AgNCs was reduced greatly, whereas tlie red fluorescence of AgNCs was increased. The concentration of HPV-16 DNA in the samples was characterized by the ratio of fluorescence intensity at 570 and 630 nm. Tlie ratiometric nanoprobes showed good selectivity for HPV-16 DNA, and the detection limit was 2 ninol/L. In addition, the practical applicability of this strategy was demonstrated by analysing the HPV-16 DNA in hiunan serum, illustrating its potential promise for clinical diagnosis.展开更多
We present here a p H-responsive activatable aptamer probe for targeted cancer imaging based on i-motif-driven conformation alteration. This p H-responsive activatable aptamer probe is composed of two single-stranded ...We present here a p H-responsive activatable aptamer probe for targeted cancer imaging based on i-motif-driven conformation alteration. This p H-responsive activatable aptamer probe is composed of two single-stranded DNA. One was used for target recognition, containing a central, target specific aptamer sequence at the 3′-end and an extension sequence at the 5′-end with 5-carboxytetramethylrhodamine(TAMRA) label(denoted as strand A). The other(strand I), being competent to work on the formation of i-motif structure, contained four stretches of the cytosine(C) rich domain and was labeled with a Black Hole Quencher 2(BHQ2) at the 3′-end. At neutral or slightly alkaline p H, strand I was hybridized to the extension sequence of strand A to form a double-stranded DNA probe, termed i-motif-based activatable aptamer probe(I-AAP). Because of proximityinduced energy transfer, the I-AAP was in a "signal off" state. The slightly acidic p H enforced the strand I to form an intramolecular i-motif and then initiated the dehybridization of I-AAP, leading to fluorescence readout in the target recognition. As a demonstration, AS1411 aptamer was used for MCF-7 cells imaging. It was displayed that the I-AAP could be carried out for target cancer cells imaging after being activated in slightly acidic environment. The applicability of I-AAP for tumor tissues imaging has been also investigated by using the isolated MCF-7 tumor tissues. These results implied the I-AAP strategy is promising as a novel approach for cancer imaging.展开更多
A novel near-infrared light photothemal-activated H2S-donating nanocomposite hydrogel was developed,through combination of a thermo-labile H2S donor and photothermal nanoparticles in agarose hydrogel.The polyethylenim...A novel near-infrared light photothemal-activated H2S-donating nanocomposite hydrogel was developed,through combination of a thermo-labile H2S donor and photothermal nanoparticles in agarose hydrogel.The polyethylenimine dithiocarbamate polymer,a thermo-labile compound,was synthesized as a novel H2S donor.The combination of a thermo-labile hydrogen sulfide donor and photothermal nanoparticles enabled the generation of H2S in agarose hydrogel upon irradiation with near-infrared light.The ability to modulate the photoirradiation for controlled generation and spatiotemporally release of H2S are its specific advantages.This photothermal spatiotemporally controlled H2S-releasing strategy was successfully applied to anti-inflammation treatment in a rat model,demonstrating its utility as a novel H2S-based therapeutic approach.展开更多
A pH-sensitive controlled release system was proposed in this work, which consists of mesoporous silica nanoparticles(MSNs) functionalized on the pore outlets with poly(4-vinylphenybronic acid-co-2-(dimethylamino...A pH-sensitive controlled release system was proposed in this work, which consists of mesoporous silica nanoparticles(MSNs) functionalized on the pore outlets with poly(4-vinylphenybronic acid-co-2-(dimethylamino)ethyl acrylate) [P(VPBA-DMAEA)]. Four kinds of P(VPBA-DMAEA)-gated MSNs were synthesized and applied for the p H-sensitive controlled release. The results showed that P(VPBADMAEA) can work as a p H-sensitive nanovalve. The release behavior of the hybrid nanoparticles could be adjusted by changing the mole ratio of VPBA and DMAEA. With the increasing of the mole ratio of VPBA,the leakage of the entrapped molecules in the pores of MSNs could be decreased at neutral and alkaline conditions. By altering the p H of buffer from 4.0 to 8.0, the valve could be switched ‘‘on'' and ‘‘off''reversibly. In addition, cells viability results indicated that these P(VPBA-DMAEA)-gated MSNs had good biocompatibility. We believe that these MSNs based p H-sensitive controlled release system will provide a promising nanodevice for sited release of drug delivery.展开更多
A novel method for the detection of PDGF-BB has been developed using double-strand DNA-copper nanoparticles (dsDNA-CuNPs) as fluorescent markers. This assay relies on the premise that the aptamer- based probe underg...A novel method for the detection of PDGF-BB has been developed using double-strand DNA-copper nanoparticles (dsDNA-CuNPs) as fluorescent markers. This assay relies on the premise that the aptamer- based probe undergoes a conformational change upon binding with target protein, and subsequently triggers polymerization reaction to generate dsDNA. Then, the resultant dsDNA can be used as a template for the formation of CuNPs with high fluorescence. Under the optimized conditions, the proposed assay allowed sensitive and selective detection of PDGF-BB with a detection limit of 4 nmol/L. This possibly makes it an attractive platform for the detection of a variety of biomolecules whose aptamers undergo similar conformational change.展开更多
A kind of temperature-sensitive nanotube array membrane was developed by modifying gold-nanotube array membranes with poly(N-isopropylacrylamide) (PNIPAm). The permeation ability of the mem-branes at different tempera...A kind of temperature-sensitive nanotube array membrane was developed by modifying gold-nanotube array membranes with poly(N-isopropylacrylamide) (PNIPAm). The permeation ability of the mem-branes at different temperatures was investigated using sodium fluorescein and quantum dots as probes. The results showed that the pore diameter of nanotube was changed due to the reversible re-sponse of PNIPAm-modified membranes to temperature, and then the permeation ability of the mem-branes was changed. The permeation of fluorescence probes was slow and even almost blocked at 25℃ (below the lower critical solution temperature, LCST), since PNIPAm formed expanded structures and decreased the pore size. While at 40℃ (above the LCST), the permeation was increased, since PNIPAm became compact structures and the pore diameter was increased. Furthermore, the permea-tion ability of the temperature-sensitive nanotube array membranes could be adjusted reversibly and it is possible to use the membranes in nanofluidic devices, nanogates, etc.展开更多
Simultaneous and quantitative detection of multiple exosomal micro RNAs(miRNAs)was successfully performed by a surface-enhanced Raman scattering(SERS)assay consisting of Raman probes and capture probes.In this design,...Simultaneous and quantitative detection of multiple exosomal micro RNAs(miRNAs)was successfully performed by a surface-enhanced Raman scattering(SERS)assay consisting of Raman probes and capture probes.In this design,the asymmetric core-shell structured Au@Au@Ag nanoparticles were first synthesized by layer-by-layer self-assembly method and modified with different Raman molecules and recognition sequences(poly A-DNA)to prepare the surface-enhanced Raman probes.Then,the streptavidinmodified magnetic beads were used to immobilize the biotinylated DNA capture sequences(biotin-DNA)to obtain capture probes.In the presence of target exosomal miRNAs,the Raman probes and capture probes could bind to the target exosomal miRNAs in the partial hybridization manner.Thus,the developed SERS sensor could indicate the target miRNAs levels in the buffer solution.Using breast cancerrelated miRNAs as model targets,the limits of detection of this sensor were determined to be 1.076 fmol/L for synthetic miR-21,0.068 fmol/L for synthetic miR-126,and 4.57 fmol/L for synthetic miR-1246,respectively.Such SERS sensors were further employed to detect the miR-21 in 20%human serum and the extraction solution of exosomes,respectively.Therefore,simultaneous and multiplex detection of cancerrelated exosomal miRNAs by this assay could provide new opportunities for further biomedical applications.展开更多
基金supported by the National Natural Science Foundation of China(22174042 and 22374038)the Natural Science Foundation for Distinguished Young Scholars of Hunan Province(2021JJ10011)the Postgraduate Scientific Research Innovation Project of Hunan Province(CX20220390)。
文摘DNAzyme-based gene therapy faces some challenges including cell penetration,activity limitation,and co-delivery functions.Self-assembled DNA nanomedicine has attracted widespread attention due to its many advantages.It is urgent to develop a universal DNA degradation strategy for precise programmable drug release.Herein,we reported a self-catabolic DNAzyme nanospheres(SCNS),which could simultaneously achieve cell penetration,activity enhancement,and co-delivery functions.The SCNS were assembled through Y-DNA stepwise hybridization with each other,which were then loaded with aptamer(Apt),doxorubicin(Dox),and zinc oxide nanoparticles(ZnO NPs).The acid-triggered dissociation of ZnO NPs leads to the generation of Zn^(2+)ions cofactors for immediately self-catabolic DNAzyme nanospheres.After the disassembly of the SCNS,three types of anticancer treatments would be activated,which include Zn^(2+)involved reactive oxygen species(ROS),Dox-induced chemotherapy,and DNAzyme-based gene therapy.The experimental results show that the nanoplatform(Apt-SCNS-Dox-ZnO)has a good tumor-killing effect and minimal side effects.As a smart self-driven drug delivery nanoplatform,it is anticipated to displace extraordinary potential in biomedicine and bioengineering.
基金supported in part by the National Key Basic Research Program of China(No.2002CB513110)Natural Science Foundation of China(No.90606003,No.20505007)+1 种基金Major International(Regional)Joint Research Program of Natural Science Foundation of China(No.20620120107)project supported by Hunan Provincial Natural Science Foundation of China(No.08JJ1002).
文摘Detection of deoxyribozyme (DNAzyme) cleavage process usually needs complex and time-consuming radial labeling, gel electrophoresis and autoradiography. This paper reported an approach to detect DNAzyme cleavage process in real time using a fluorescence probe. The probe was employed as DNAzyme substrate to convert directly the cleavage information into fluorescence signal in real time. Compared with traditional approach, this non-isotope method not only brought a convenient means to monitor the DNAzyme cleavage reaction, but also offered abundant dynamic data for choosing potential gene therapeutic agents. It provides a new tool for DNAzyme research, as well as a new insight into research on human disease diagnosis. Based on this method, 8- 17deoxyribozyme (8-17DNAzyme) against hepatitis C virus RNA (HCV-RNA) was designed and the cleavage process was studied in real time. ?2009 Ke Min Wang. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All fights reserved.
基金the National Key Basic Research Program(Grant No.2002CB513110)the Key Technologies Research Development Program of China(Grant Nos.2003BA310A16and2005EP090026)+2 种基金International Technologies Collaboration Program of China(Grant No.2003DF000039)the National Natural Science Foun-dation of China(Grant Nos.90606003and20475015)Key Project of Hunan Province Technology Plan of China(Grant No.0399Y1006)
文摘Studying the expression level of mRNA in living cells will offer tremendous opportunities for ad-vancement in cell biology research,disease diagnostics,and drug discovery.In this paper,a molecular beacon(MB)specific for the important tumor suppressor gene p21 has been designed and synthesized.The fluorescence signal was detected in real-time after the MB entered the cytoplasm of nasopharyn-geal carcinoma cells.After injecting the p21MB into nasopharyngeal carcinoma cell and p33-trans-fected nasopharyngeal carcinoma cell,the consistent increase of fluorescent signal intensity was de-tected in both cell lines,and maximum fluorescence intensity achieved in about 15 min.In about 4 min following microinjection,the fluorescence increasing rate was significantly different between these two cell lines,which indicate the different p21 mRNA expression levels.The results obtained in the real-time detection were also validated by RT-PCR.Analysis of the initial fluorescence increasing rate can effi-ciently reduce the side effect of enzyme and improve the accuracy in living cell mRNA detection.
基金Supported by the National Key Basic Research Program (Grant No. 2002CB513110)International Technologies Collaboration Program of China (Grant No. 2003DF000039)National Natural Science Foundation of China (Grant Nos. 90606003 and 20620120107)
文摘In this study, GFP mRNA in COS-7 cell and GFP-transfected COS-7 cell was detected in real time using phosphorothioate-modified molecular beacon based on living cell imaging method. Results showed that phosphorothioate-modified molecular beacon still kept the advantages of molecular beacon, such as, excellent selectivity, high sensitivity, and no separation detection. In addition, this modification could significantly increase the nuclease resistance of molecular beacon. Phosphorothioate-modified molecular beacon can efficiently reduce the false positive signal and improve the accuracy of living cell mRNA detection.
基金the financial support of the National Natural Science Foundation of China (Nos.21190044,21205032,and 91027000)National Natural Science Foundation of Postdoctoral Scientists of China (No.2013M531779)+1 种基金Hunan Provincial Natural Science Foundation of China (No.13JJ4032)the Fundamental Research Funds for the Central Universities of China.
文摘Combining the inhibited aptazyme and molecular beacon (MB), we developed a versatile sensing strategy for amplified detection of adenosine. In this strategy, the adenosine aptamer links to the 8-17 DNAzyme to form an aptazyme. A short sequence, denoted as inhibitor, is designed to form a duplex spanning the aptamer-DNAzyme junction, which blocks the catalytic function of the DNAzyme. Only in the presence of target adenosine, the aptamer binds to adenosine, thus the inhibitor dissociates from the aptamer portion of the aptazyme and can no longer form the stable duplex required to inhibit the catalytic activity of the aptazyme. The released DNAzyme domain will hybridize to the MB and catalyze the cleavage in the presence of Zn2+, making the fluorophore separate from the quencher and resulting in fluorescence signal. The results showed that the detection method has a dynamic range from 10 nmol/L to 1nmol/L, with a detection limit of 10 nmol/L.
基金supported by the National Natural Science Foundation of China(Nos.21190044,21175035)National Basic Research Program(No.2011CB911002)International Science & Technology operation Program of China(No.2010DFB30300)
文摘A one-step and facile strategy toward the construction of multicolor polymers via supramolecular selfassembly was proposed.Multicolor polymers were simply prepared by the self-assembly of adamantane-labeled fluorescein,adamantane-labeled rhodamine B and β-cyclodextrin polymers via host-guest interaction between β-cyclodextrin and adamantane.Multicolor polymers showed several interesting properties:multiple emission signatures by a single wavelength excitation,easy tunability,intense fluorescence,high photostablility.In addition,the self-assembly approach implied a facile and flexible strategy for constructing functionalized materials,such as multicolor materials for biological labeling and imaging,and sensing materials for the detection of physiological parameters.
基金supported by the National Natural Science Foundation of China(21735002,22177032,32101082)the Science Fund for Distinguished Young Scholars of Hunan Province(2021JJ10013)+1 种基金the Hunan Province Innovative Talent Funding for Postdoctoral Fellows(2021RC2059)the Postdoctoral Science Foundation of China(2021TQ0103,2021M690957).
文摘Implanting artificial organelles in living cells is capable of correcting cellular dysfunctionalities for cell repair and biomedical applications. In this work, phase-separated bienzyme-loaded coacervate microdroplets are established as a model of artificial membraneless organelles in endothelial dysfunctional cells for the cascade enzymatic production of nitric oxide(NO) with a purpose of correcting cellular NO deficiency. We prepared the coacervate microdroplets via liquid-liquid phase separation of oppositely charged polyelectrolytes, in which glucose oxidase/horseradish peroxidase-mediated cascade reaction was compartmented. After the coacervate microdroplets were implanted in NO-deficient dysfunctional cells, the compartments maintained a phase-separated liquid droplet structure, which facilitated a significant enhancement of NO production in the dysfunctional cells. The recovery of NO production was further exploited to inhibit clot formation in blood plasma located in the cell suspension. This demonstrated a proof-of-concept design of artificial organelles in dysfunctional cells for cell repair and anticoagulation-related medical applications. Our results demonstrate an approach for the construction of coacervate droplets through phase separation for the generation of artificial membraneless organelles, which can be designed to provide an array of functionalities in living organisms that have the potential to be used in the field of cell engineering and medical therapy.
基金supported by the Natural Science Foundation of China(Nos.21877030,21735002,21778016 and 21521063)。
文摘Accurate detection and imaging of adenosine triphosphate(ATP)expression levels in living cells is of great value for understanding cell metabolism,physiological activities,and pathologic mechanisms.Here,we developed a DNA tetrahedron-based split aptamer probe(TD probe)for ratiometric fluorescence imaging of ATP in living cells.The TD probe is constructed by hybridizing two split ATP aptamer probes(Apt-a and Apt-b)to a DNA tetrahedron assembled by four DNA oligonucleotides(T1,T2,T3 and T4).In the presence of ATP,the TD probe will alter its structure from the open to closed state,thus bringing the separated donor and acceptor fluorophores into close proximity for high fluorescence resonance energy transfer(FRET)signals.The TD probe exhibits low cytotoxicity,efficient cell internalization and good biological stability.Moreover,based on the FRET“off”to“on”signal output mode,the TD probe can effectively avoid false-positive signals from complex biological matrices,which is significant for long-term reliable imaging in living cells.In addition,by changing the split aptamers attached to DNA tetrahedron,the proposed strategy may be extended for detecting various intracellular targets.Collectively,this strategy provides a valuable sensing platform for biomarkers analysis in living cells,thus having great potential for early clinical diagnosis and therapeutic evaluation.
基金supported by the Natural Science Foundation of China(Nos.21877030,21735002 and 21778016).
文摘Accurate and sensitive detection of caner cells is of significant importance for early diagnosis and treat-ment of cancer.Here,we developed an extracellular ATP-dctivated hybridization chain reaction(HCR)amplification strategy to meet this purpose.This strategy relies on three DNA probes,Apt-trigger,H1-AТP aptamer duplex and hairpin H2.The Apt-trigger probe consists of two com sequence for specific recognition of the target cells.and a trigger sequence for the HCR assembly.Theроnents:an aptamer duplex structure of H1-ATP aptamer causes the tochold in hairpin H1 to be hidden,preventing the strand-ent displacement reaction between haipin H1 and Apt-trigger.Upon activation with ATP the ATP aptamer will blnd to ATP to dissoci iate from hairpin H1,thus leading to an Apt-trigger-induced strand-displacement reaction and subsequent HCR with hairpin H2 on the target cell surface.Benefiting from aptamer recogni-tion and ATP-activated HCR amplification,this strategy can not only perform sensitive quantitative anal-ysis with a detection limit of 25 cells in 200 ul.of binding buffer,but also show desirable specificity and accuracy for identifylng target cells from control cells and mixed cell samples,Imporantly,this method retains stable and good perfor mance for target cell detection in 10%fetal bovine ser rum,den onstrating great potential for clinical diagnosis in complex biological matrices.Furthermore,this strategy can be adapted to detect various types of cancer cells by changing the corresponding aptamer sequence.
基金the National Key Basic Research Program of China (Grant No. 2002CB513110)Key Technologies Research and Development Program of China (Grant No. 2003BA310A16 and 2005EP090026)+1 种基金Key Project of International Technologies Collaboration Program of China (Grant No. 2003DF000039)the National Natural Science Foundation of China (Grant No. 90606003, 20475015 and 20620120107)
文摘In recent years, specific detection of proteins is one of the hot issues about aptamers in proteomics. Here we reported a simple, sensitive and specific proximity-dependent protein assay with dual DNA aptamers. Thrombin was used as the model protein, and two aptamer probes with complementary se- quence at 3′-end were designed for the two distinct epitopes of the protein. Association of the two ap- tamers with thrombin resulted in stable hybrids due to the proximity of 3′-end, then polymerase reac- tion was induced. The amount of obtained dsDNA was indicated using the fluorescence dye Sybr Green I. The results showed that the initial velocity of polymerase reaction had a positive correlation with concentration of thrombin. The advantages of this dual-aptamer-based approach included simple and flexible design of aptamer probes, high selectivity and high sensitivity. The detection limit was 6.9 pmol/L.
基金supported in part by the Key Project of Natural Science Foundation of China (Nos.21775036,21675046,21735002, 21521063 and 21874035)the Key Point Research and Invention Program of Hunan Province (No.2017DK2011)+1 种基金the Research Foundation of Education Bureau of Hunan Province (No.18B027)the Hunan Provincial Natural Science Foundation (No.2018JJ2033)
文摘In clinical cancer research,it is quite promising to develop multimodal synergistic therapeutic strategies.Photodynamic and photothermal synergistic therapy is a very desirable multimodal therapy strategy.Herein,we report a facile and simple method to construct a nanotherapeutic agent for photodynamic and photothermal therapy.This nanotherapeutic agent(ZnO@Ce6-PDA)is composed of a ZnO nanoparticle core,an interlayer of photosensitizer chlorin e6(Ce6)and an outer layer of polydopamine(PDA).Due to the existence of Ce6,the ZnO@Ce6-PDA can efficiently generate singlet oxygen(1O2)under 660 nm laser irradiation.Moreover,the ZnO@Ce6-PDA can serve as a photothermal agent,because of the excellent photothermal conversion efficiency of the PDA coating layer in the presence of 780 nm laser.Experiment results demonstrated that the designed nanotherapeutic agent had outstanding phototoxicity upon the combination of laser irradiation at 660 and 780 nm.Thus,our work proves that the ZnO@Ce6-PDA is a promising photodynamic/photothermal dual-modal nanotherapeutic agent for enhanced cancer therapy.
基金supported by the National Natural Science Foundation of China(Nos.21190044 and 21175035)National Basic Research Program(No.2011CB911002)International Science&Technology operation Program of China(No.2010DFB30300)
文摘This work develops a fluorescence approach for sensitive detection of DNA methyltransferase activity based on endonuclease and rolling circle amplification (RCA) technique. In the presence of DNA adenine methylation (Dam) MTase, the methylation-responsive sequence of hairpin probe is methylated and cleaved by the methylation-sensitive restriction endonuclease Dpn 1. The products cleaved by restriction endonuclease Dpn I then function as a signal primer to initiate RCA reaction by hybridizing with the circular DNA template. Each RCA product containing thousands of repeated sequences might hybridize with a large number of molecular beacons (detection probes), resulting in an enhanced fluorescence signal. In the absence of Dam MTase, neither methylation/cleavage nor RCA reaction can be initiated and no fluorescence signal is observed. The proposed method exhibits a dynamic range from 0.5 U/mL to 30 U/mL and a detection limit of 0.18 U/mL. This method can be used for the screening of antimicrobial drugs and has a great potential to be further applied in early clinical diagnosis.
基金the financial support from the National Natural Science Foundation of China(Nos. 21675047, 21735002 and 21521063)the Key Point Research and Invention Program of Hunan Province(No. 2017DK2011)
文摘Biomimetic nanodevice is an important branch to expand the potential applications of artificial nanochannels. Here, we constructed nanochannels for cascade response of borate and cis-hydroxyl compounds by modifying the nanochannels of track-etched polycarbonate (TEPC) membranes with polyvinyl alcohol (PVA). Firstly, borate bound to PVA and increased the negative charge density on the surface of the nanochannels, which obstructed the transport of 1,5-naphthalene disulfonate (NDS2-). Subsequently, the addition of cis-hydroxyl compounds induced leaving of borate due to the stronger binding affinities between borate and cis-hydroxyl compounds, which reduced the negative charge density on nanochannels and then enhanced the transport of NDS2-. The cascade response of the nanochannels also accord with the properties of IMP (implication) logic gate. In addition, such nanochannels showed good reproducibility and reversibility. Therefore, this cascade response model based on nanochannels has the potential to be used as switches in area of actuators and biosensors, and is also expected to be used to understand the interaction of substances in nanoscale and simulate the physiological functions of boron.
基金Supported by the National Natural Science Foundation of China(Nos.21675047, 21735002, 21521063)the Key Point Research and Invention Program of Hunan Province, China(No.2017DK2011).
文摘Herein, we described a ratiometric strategy based on "chameleon" DNA-silver nanoclusters( DNA-AgNCs) fluorescent binary probes. The strategy was applied to detect high-risk human papillomavirus( HPV) DNA sequences, HPV-16. First, DNA-AgNCs were synthesized by a simple reduction method. The obtained nanoprobes showed typical yellow and red fluorescence of AgNCs. Upon the addition of HPV-16 DNA, the yellow fluorescence of AgNCs was reduced greatly, whereas tlie red fluorescence of AgNCs was increased. The concentration of HPV-16 DNA in the samples was characterized by the ratio of fluorescence intensity at 570 and 630 nm. Tlie ratiometric nanoprobes showed good selectivity for HPV-16 DNA, and the detection limit was 2 ninol/L. In addition, the practical applicability of this strategy was demonstrated by analysing the HPV-16 DNA in hiunan serum, illustrating its potential promise for clinical diagnosis.
基金supported by the Key Project of National Natural Science Foundation of China (21175039, 21322509, 21305035, 21190044, 21221003, 21305038, 2015JJ3044)
文摘We present here a p H-responsive activatable aptamer probe for targeted cancer imaging based on i-motif-driven conformation alteration. This p H-responsive activatable aptamer probe is composed of two single-stranded DNA. One was used for target recognition, containing a central, target specific aptamer sequence at the 3′-end and an extension sequence at the 5′-end with 5-carboxytetramethylrhodamine(TAMRA) label(denoted as strand A). The other(strand I), being competent to work on the formation of i-motif structure, contained four stretches of the cytosine(C) rich domain and was labeled with a Black Hole Quencher 2(BHQ2) at the 3′-end. At neutral or slightly alkaline p H, strand I was hybridized to the extension sequence of strand A to form a double-stranded DNA probe, termed i-motif-based activatable aptamer probe(I-AAP). Because of proximityinduced energy transfer, the I-AAP was in a "signal off" state. The slightly acidic p H enforced the strand I to form an intramolecular i-motif and then initiated the dehybridization of I-AAP, leading to fluorescence readout in the target recognition. As a demonstration, AS1411 aptamer was used for MCF-7 cells imaging. It was displayed that the I-AAP could be carried out for target cancer cells imaging after being activated in slightly acidic environment. The applicability of I-AAP for tumor tissues imaging has been also investigated by using the isolated MCF-7 tumor tissues. These results implied the I-AAP strategy is promising as a novel approach for cancer imaging.
基金financial support of the National Natural Science Foundation of China(Nos.21735002,21575037,21778016,21675046,21877030)Natural Science Foundation of Hunan Province(No.2177JJ3026)Keypoint Research and Invention Program of Hunan Province(No.2017DK2011)。
文摘A novel near-infrared light photothemal-activated H2S-donating nanocomposite hydrogel was developed,through combination of a thermo-labile H2S donor and photothermal nanoparticles in agarose hydrogel.The polyethylenimine dithiocarbamate polymer,a thermo-labile compound,was synthesized as a novel H2S donor.The combination of a thermo-labile hydrogen sulfide donor and photothermal nanoparticles enabled the generation of H2S in agarose hydrogel upon irradiation with near-infrared light.The ability to modulate the photoirradiation for controlled generation and spatiotemporally release of H2S are its specific advantages.This photothermal spatiotemporally controlled H2S-releasing strategy was successfully applied to anti-inflammation treatment in a rat model,demonstrating its utility as a novel H2S-based therapeutic approach.
基金supported by the National Natural Science Foundation of China (Nos. 21190040, 21175035, 21375034)National Basic Research Program of China (No. 2011CB911002)International Science & Technology Cooperation Program of China (No. 2010DFB30300)
文摘A pH-sensitive controlled release system was proposed in this work, which consists of mesoporous silica nanoparticles(MSNs) functionalized on the pore outlets with poly(4-vinylphenybronic acid-co-2-(dimethylamino)ethyl acrylate) [P(VPBA-DMAEA)]. Four kinds of P(VPBA-DMAEA)-gated MSNs were synthesized and applied for the p H-sensitive controlled release. The results showed that P(VPBADMAEA) can work as a p H-sensitive nanovalve. The release behavior of the hybrid nanoparticles could be adjusted by changing the mole ratio of VPBA and DMAEA. With the increasing of the mole ratio of VPBA,the leakage of the entrapped molecules in the pores of MSNs could be decreased at neutral and alkaline conditions. By altering the p H of buffer from 4.0 to 8.0, the valve could be switched ‘‘on'' and ‘‘off''reversibly. In addition, cells viability results indicated that these P(VPBA-DMAEA)-gated MSNs had good biocompatibility. We believe that these MSNs based p H-sensitive controlled release system will provide a promising nanodevice for sited release of drug delivery.
基金supported by the National Natural Science Foundation of China(Nos.21190041,21190044,21175035)National Basic Research Program(No.2011CB911002)Interna-tional Science&Technology operation Program of China(No.2010DFB30300)
文摘A novel method for the detection of PDGF-BB has been developed using double-strand DNA-copper nanoparticles (dsDNA-CuNPs) as fluorescent markers. This assay relies on the premise that the aptamer- based probe undergoes a conformational change upon binding with target protein, and subsequently triggers polymerization reaction to generate dsDNA. Then, the resultant dsDNA can be used as a template for the formation of CuNPs with high fluorescence. Under the optimized conditions, the proposed assay allowed sensitive and selective detection of PDGF-BB with a detection limit of 4 nmol/L. This possibly makes it an attractive platform for the detection of a variety of biomolecules whose aptamers undergo similar conformational change.
基金Supported by the National Key Basic Research Program of China (Grant No. 2002CB513110)Key Technologies Research and Development Program of China (Grant Nos. 2003BA310A16 and 2005EP090026)+1 种基金Key Project of International Technologies Collaboration Program of China (Grant No. 2003DF000039)National Natural Science Foundation of China (Grant Nos. 90606003, 20475015 and 20620120107)
文摘A kind of temperature-sensitive nanotube array membrane was developed by modifying gold-nanotube array membranes with poly(N-isopropylacrylamide) (PNIPAm). The permeation ability of the mem-branes at different temperatures was investigated using sodium fluorescein and quantum dots as probes. The results showed that the pore diameter of nanotube was changed due to the reversible re-sponse of PNIPAm-modified membranes to temperature, and then the permeation ability of the mem-branes was changed. The permeation of fluorescence probes was slow and even almost blocked at 25℃ (below the lower critical solution temperature, LCST), since PNIPAm formed expanded structures and decreased the pore size. While at 40℃ (above the LCST), the permeation was increased, since PNIPAm became compact structures and the pore diameter was increased. Furthermore, the permea-tion ability of the temperature-sensitive nanotube array membranes could be adjusted reversibly and it is possible to use the membranes in nanofluidic devices, nanogates, etc.
基金the Project of National Natural Science Foundation of China(Nos.21775036,21735002,21874035 and 22174044)。
文摘Simultaneous and quantitative detection of multiple exosomal micro RNAs(miRNAs)was successfully performed by a surface-enhanced Raman scattering(SERS)assay consisting of Raman probes and capture probes.In this design,the asymmetric core-shell structured Au@Au@Ag nanoparticles were first synthesized by layer-by-layer self-assembly method and modified with different Raman molecules and recognition sequences(poly A-DNA)to prepare the surface-enhanced Raman probes.Then,the streptavidinmodified magnetic beads were used to immobilize the biotinylated DNA capture sequences(biotin-DNA)to obtain capture probes.In the presence of target exosomal miRNAs,the Raman probes and capture probes could bind to the target exosomal miRNAs in the partial hybridization manner.Thus,the developed SERS sensor could indicate the target miRNAs levels in the buffer solution.Using breast cancerrelated miRNAs as model targets,the limits of detection of this sensor were determined to be 1.076 fmol/L for synthetic miR-21,0.068 fmol/L for synthetic miR-126,and 4.57 fmol/L for synthetic miR-1246,respectively.Such SERS sensors were further employed to detect the miR-21 in 20%human serum and the extraction solution of exosomes,respectively.Therefore,simultaneous and multiplex detection of cancerrelated exosomal miRNAs by this assay could provide new opportunities for further biomedical applications.