AIM: To evaluate the inhibitory effects of DNAzymes on the expressions of hepatitis B virus (HBV) s (HBsAg) and e (HBeAg) in 2.2.15 cells, and to explore the potential therapeutic effects of DNAzymes on replication of...AIM: To evaluate the inhibitory effects of DNAzymes on the expressions of hepatitis B virus (HBV) s (HBsAg) and e (HBeAg) in 2.2.15 cells, and to explore the potential therapeutic effects of DNAzymes on replication of HBV genome. METHODS: DNAzymes DrzBS and DrzBC specific to HBV (aywsubtype) s gene ORF A^157UG and e gene ORF A^1816UG, were designed and synthesized. Inhibitory effects of DrzBS or DrzBC on the expressions of HBV s and e genes as well as HBV DNA levels in culture supernatants were observed in 2.2.15 cells. RESULTS: After being treated with DrzBS or DrzBC, the expression of HBV s or e genes in 2.2.15 cells was depressed dramatically. The maximum inhibition rate was 94.2% and 91.8% for DrzBS and DrzBC, respectively. The concentration for effective inhibition of both DrzBS and DrzBC was within 0.1-2.5 μmol/L, showing a dosedependence. The efficiency of inhibiting HBsAg, HBeAg in 2.2.15 cells by DrzBS or DrzBC was higher than that of the same target genes by antisense oligonucleotides (ASON). The concentration for effective inhibition of DNAzymes was at least 10-fold lower compared with ASON controls. Neither inhibition on the replication of HBV DNA nor toxicity to 2.2.15 cells was observed. CONCLUSION: DrzBS and DrzBC can block the expression of HBV s- and e-genes in 2.2.15 cells and provide a specific and effective anti-HBV gene therapeutic means.展开更多
In molecular engineering,designing and synthesizing molecular machines with capable of performing complex tasks,remains a formidable challenge.DNA is an excellent candidate for building molecular robots because it is ...In molecular engineering,designing and synthesizing molecular machines with capable of performing complex tasks,remains a formidable challenge.DNA is an excellent candidate for building molecular robots because it is highly programmable.Here,we present an artificial nanorobot,in which a DNA cube serves as the inert‘body’,and nucleic acid catalysts based on an enzymatic nicking reaction act as the‘legs’for walking.The nanorobot can execute a series of actions,such as‘start’,‘turn’,and‘stop’when it walks along a predefined track.Its performance could be confirmed and monitored by using an atomic force microscope(AFM)and fluorescence spectroscopy.Inspired by biological machines,we artificially designed a series of specialized tasks that combined walking with control of cargo transport and catalysis.Real-time fluorescence kinetics curves provide monitoring signals for cargo transport and catalytic processes.Our work can enrich the toolbox of DNA machinery and has great potential for engineering molecular nanofactories.展开更多
The uranyl ion(UO22+) poses high risks to human health and the environment, hence its detection and monitoring is of utmost significance. However, the development of an ultra-sensitive, high-efficiency and convenient ...The uranyl ion(UO22+) poses high risks to human health and the environment, hence its detection and monitoring is of utmost significance. However, the development of an ultra-sensitive, high-efficiency and convenient approach for on-site detection of UO22+ remains a challenge. Herein, a reliable and reusable surface-enhanced Raman spectroscopy(SERS)-based microfluidic biosensor was developed for rapid detection of UO22+ in real samples. The detection protocol involved the reaction of 5′-Rhodamine B(RhB)-labeled double-stranded DNA for UO22+-specific DNAzyme-cleavage reaction in a U-shaped microchannel. Then, the reaction products were delivered into three parallel samples for high-throughput tests by SERS biochips,where 3 D ZnO-Ag mesoporous nanosheet arrays(MNSs) were modified with a single-stranded DNA(ssDNA). The ssDNAwas sequence-complementary with the 5′-RhB-labeled cleaved-stranded DNA(csDNA) from the reaction products. By the hybridization of ssDNA and csDNA, the signal probe RhB was fixed close to the surface of the ZnO-Ag MNSs to enhance the Raman signal. The limit of detection for UO22+ with the microfluidic-SERS biosensor was 3.71×10-15 M. An over 20,000-fold selectivity towards UO22+ response was also achieved in the presence of 15 other metal ions. The high-throughput microfluidicSERS biosensor operated well for practical UO22+ detection, with excellent recoveries in contaminated river and tap water from95.2% to 106.3%(relative standard deviation(RSD)<6.0%, n=6). Although the SERS-based microfluidic biosensor developed in this study was deployed for the detection of UO22+, the reusable and high-efficiency system may be expanded to the detection of other analytes on-site.展开更多
A label-free deoxyribozyme(DNAzyme)biosensor for m^(6) demethylase activity detection is developed.When demethylase FTO(Fat mass and obesity-associated protein),an important m^(6) demethylase of ALKBH demethylase fami...A label-free deoxyribozyme(DNAzyme)biosensor for m^(6) demethylase activity detection is developed.When demethylase FTO(Fat mass and obesity-associated protein),an important m^(6) demethylase of ALKBH demethylase family is present,the m^(6) group is specifically demethylated,and the cleavage activity of DNAzyme is restored.This allows the cleavage product that contains G-quadruplex sequence to bind to thioflavin T(ThT)and generate fluorescence signals.The biosensor shows high specificity and sensitivity,and fast reaction speed.Our study demonstrates a new design of allosteric DNAzyme for sensing.This method represents the first label-free nucleic acid biosensor for FTO assay,providing a feasible route towards inhibitors screening.展开更多
Mercury ion(Hg^(2+)),a highly noxious of heavy metalion,has detrimental effects on the ecological environment and human health.Herein,we have developed an exonuclease III(Exo III)assisted catalytic hairpin assembly fo...Mercury ion(Hg^(2+)),a highly noxious of heavy metalion,has detrimental effects on the ecological environment and human health.Herein,we have developed an exonuclease III(Exo III)assisted catalytic hairpin assembly formation of a trivalent G-quadruplex/hemin DNAzyme for colorimetric detection of Hg^(2+).A hairpin DNA(Hr)was designed with thymine-Hg^(2+)-thymine pairs that catalyzed by Exo III is prompted to happen upon binding Hg^(2+).A released DNA fragment triggers the catalytic assembly of other three hairpins(H1,H2,and H3)to form many trivalent G-quadruplex/hemin DNA enzymes for signal output.The developed sensor shows a dynamic range from 2 pM to 2μM,with an impressively low detection limit of 0.32 pM for Hg^(2+)detection.Such a sensor also has good selectivity toward Hg^(2+)detection in the presence of other common metal ions.This strategy shows the great potential for visual detection with portable type.展开更多
脱氧核酶(DNAzyme)是通过体外筛选技术(systematic evolution of ligands by exponential enrichment,SELEX技术)得到的具有酶活性的单链DNA片段。与天然酶相比,脱氧核酶具有性质稳定、合成和修饰简单以及易于储存等优势。某些脱氧核酶...脱氧核酶(DNAzyme)是通过体外筛选技术(systematic evolution of ligands by exponential enrichment,SELEX技术)得到的具有酶活性的单链DNA片段。与天然酶相比,脱氧核酶具有性质稳定、合成和修饰简单以及易于储存等优势。某些脱氧核酶对金属离子显示了高度的识别特异性,且酶活性与特定金属离子的浓度密切相关。这些特点使其在金属离子检测中的应用备受关注。本文对脱氧核酶在重金属离子传感器设计中的应用进行了总结和评述,重点讨论了荧光传感器和比色传感器的设计。展开更多
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
基金Supported by the National Natural Science Foundation of China,No.30271183
文摘AIM: To evaluate the inhibitory effects of DNAzymes on the expressions of hepatitis B virus (HBV) s (HBsAg) and e (HBeAg) in 2.2.15 cells, and to explore the potential therapeutic effects of DNAzymes on replication of HBV genome. METHODS: DNAzymes DrzBS and DrzBC specific to HBV (aywsubtype) s gene ORF A^157UG and e gene ORF A^1816UG, were designed and synthesized. Inhibitory effects of DrzBS or DrzBC on the expressions of HBV s and e genes as well as HBV DNA levels in culture supernatants were observed in 2.2.15 cells. RESULTS: After being treated with DrzBS or DrzBC, the expression of HBV s or e genes in 2.2.15 cells was depressed dramatically. The maximum inhibition rate was 94.2% and 91.8% for DrzBS and DrzBC, respectively. The concentration for effective inhibition of both DrzBS and DrzBC was within 0.1-2.5 μmol/L, showing a dosedependence. The efficiency of inhibiting HBsAg, HBeAg in 2.2.15 cells by DrzBS or DrzBC was higher than that of the same target genes by antisense oligonucleotides (ASON). The concentration for effective inhibition of DNAzymes was at least 10-fold lower compared with ASON controls. Neither inhibition on the replication of HBV DNA nor toxicity to 2.2.15 cells was observed. CONCLUSION: DrzBS and DrzBC can block the expression of HBV s- and e-genes in 2.2.15 cells and provide a specific and effective anti-HBV gene therapeutic means.
基金supported by the National Natural Science Foundation of China(Nos.21991080,22374042)the Science and Technology Major Project of Hunan Province,China(No.2021SK1020).
文摘In molecular engineering,designing and synthesizing molecular machines with capable of performing complex tasks,remains a formidable challenge.DNA is an excellent candidate for building molecular robots because it is highly programmable.Here,we present an artificial nanorobot,in which a DNA cube serves as the inert‘body’,and nucleic acid catalysts based on an enzymatic nicking reaction act as the‘legs’for walking.The nanorobot can execute a series of actions,such as‘start’,‘turn’,and‘stop’when it walks along a predefined track.Its performance could be confirmed and monitored by using an atomic force microscope(AFM)and fluorescence spectroscopy.Inspired by biological machines,we artificially designed a series of specialized tasks that combined walking with control of cargo transport and catalysis.Real-time fluorescence kinetics curves provide monitoring signals for cargo transport and catalytic processes.Our work can enrich the toolbox of DNA machinery and has great potential for engineering molecular nanofactories.
基金supported by the Science Challenge Project (TZ2018004)the National Natural Science Foundation of China (21502179)
文摘The uranyl ion(UO22+) poses high risks to human health and the environment, hence its detection and monitoring is of utmost significance. However, the development of an ultra-sensitive, high-efficiency and convenient approach for on-site detection of UO22+ remains a challenge. Herein, a reliable and reusable surface-enhanced Raman spectroscopy(SERS)-based microfluidic biosensor was developed for rapid detection of UO22+ in real samples. The detection protocol involved the reaction of 5′-Rhodamine B(RhB)-labeled double-stranded DNA for UO22+-specific DNAzyme-cleavage reaction in a U-shaped microchannel. Then, the reaction products were delivered into three parallel samples for high-throughput tests by SERS biochips,where 3 D ZnO-Ag mesoporous nanosheet arrays(MNSs) were modified with a single-stranded DNA(ssDNA). The ssDNAwas sequence-complementary with the 5′-RhB-labeled cleaved-stranded DNA(csDNA) from the reaction products. By the hybridization of ssDNA and csDNA, the signal probe RhB was fixed close to the surface of the ZnO-Ag MNSs to enhance the Raman signal. The limit of detection for UO22+ with the microfluidic-SERS biosensor was 3.71×10-15 M. An over 20,000-fold selectivity towards UO22+ response was also achieved in the presence of 15 other metal ions. The high-throughput microfluidicSERS biosensor operated well for practical UO22+ detection, with excellent recoveries in contaminated river and tap water from95.2% to 106.3%(relative standard deviation(RSD)<6.0%, n=6). Although the SERS-based microfluidic biosensor developed in this study was deployed for the detection of UO22+, the reusable and high-efficiency system may be expanded to the detection of other analytes on-site.
基金supported by the Natural Science Foundation of Jiangsu Province(BK20221258)the National Natural Science Foundation of China(22274121 and 22274123).We thank Prof.Xiang Zhou at Wuhan University for CD measurement.
文摘A label-free deoxyribozyme(DNAzyme)biosensor for m^(6) demethylase activity detection is developed.When demethylase FTO(Fat mass and obesity-associated protein),an important m^(6) demethylase of ALKBH demethylase family is present,the m^(6) group is specifically demethylated,and the cleavage activity of DNAzyme is restored.This allows the cleavage product that contains G-quadruplex sequence to bind to thioflavin T(ThT)and generate fluorescence signals.The biosensor shows high specificity and sensitivity,and fast reaction speed.Our study demonstrates a new design of allosteric DNAzyme for sensing.This method represents the first label-free nucleic acid biosensor for FTO assay,providing a feasible route towards inhibitors screening.
基金Supported by The Science and Technology Project of General Administration of Quality Supervision,Inspection and Quarantine (2015IK126)The Science and Technology Project of Changsha City of Hunan Province of China (KQ1602124).
文摘Mercury ion(Hg^(2+)),a highly noxious of heavy metalion,has detrimental effects on the ecological environment and human health.Herein,we have developed an exonuclease III(Exo III)assisted catalytic hairpin assembly formation of a trivalent G-quadruplex/hemin DNAzyme for colorimetric detection of Hg^(2+).A hairpin DNA(Hr)was designed with thymine-Hg^(2+)-thymine pairs that catalyzed by Exo III is prompted to happen upon binding Hg^(2+).A released DNA fragment triggers the catalytic assembly of other three hairpins(H1,H2,and H3)to form many trivalent G-quadruplex/hemin DNA enzymes for signal output.The developed sensor shows a dynamic range from 2 pM to 2μM,with an impressively low detection limit of 0.32 pM for Hg^(2+)detection.Such a sensor also has good selectivity toward Hg^(2+)detection in the presence of other common metal ions.This strategy shows the great potential for visual detection with portable type.
文摘脱氧核酶(DNAzyme)是通过体外筛选技术(systematic evolution of ligands by exponential enrichment,SELEX技术)得到的具有酶活性的单链DNA片段。与天然酶相比,脱氧核酶具有性质稳定、合成和修饰简单以及易于储存等优势。某些脱氧核酶对金属离子显示了高度的识别特异性,且酶活性与特定金属离子的浓度密切相关。这些特点使其在金属离子检测中的应用备受关注。本文对脱氧核酶在重金属离子传感器设计中的应用进行了总结和评述,重点讨论了荧光传感器和比色传感器的设计。
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.
