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
DNAzyme machines play critical roles in the fields of cell imaging,disease diagnosis,and cancer therapy.However,the applications of DNAzyme machines are limited by the nucleases-induced degradation,non-specific bindin...DNAzyme machines play critical roles in the fields of cell imaging,disease diagnosis,and cancer therapy.However,the applications of DNAzyme machines are limited by the nucleases-induced degradation,non-specific binding of proteins,and insufficient provision of cofactors.Herein,protected DNAzyme machines with different cofactor designs(referred to as Pro Ds)were nanoengineered by the construction of multifunctional metal-phenolic nanoshells to deactivate the interferential proteins,including nucleases and non-specific binding proteins.Moreover,the nanoshells not only facilitate the cellular internalization of Pro Ds but provide specific metal ions acting as cofactors of the designed DNAzymes.Cellular imaging results demonstrated that Pro Ds could effectively and simultaneously monitor multiple tumor-related micro RNAs in living cells.This facile and rapid strategy that encapsulates DNAzyme machines into the protective metal-phenolic nanoshells is anticipated to extend to a wide range of functional nucleic acidsbased biomedical applications.展开更多
DNA-functionalized gold nanoparticles are one of the most versatile bionanomaterials for biomedical and clinical diagnosis. Herein, we discovered that the performance of DNAzyme cleaving the substrate is highly relate...DNA-functionalized gold nanoparticles are one of the most versatile bionanomaterials for biomedical and clinical diagnosis. Herein, we discovered that the performance of DNAzyme cleaving the substrate is highly related to its length. This intriguing phenomenon only appears at the interfaces of DNAfunctionalized gold nanoparticles. We systematically investigated the causes of this phenomenon. We conjectured that the DNAzyme with extended nucleotides that do not match its substrate strand is vulnerable to non-specific adsorption, electrostatic repulsion, and steric hindrance. Based on our improved understanding of this phenomenon, we have successfully developed a highly sensitive and specific amplifiable biosensor to detect human apurinic/apyrimidinic endonuclease 1.展开更多
DNAzyme amplifiers have been extensively explored as a useful sensing platform,but single DNAzyme amplifier is limited in biosensing applications by its low sensitivity.Herein,a cascade DNAzyme amplifier was designed ...DNAzyme amplifiers have been extensively explored as a useful sensing platform,but single DNAzyme amplifier is limited in biosensing applications by its low sensitivity.Herein,a cascade DNAzyme amplifier was designed by exploiting concurrent amplification cycle principles of toehold-mediated strand displacement reaction(TSDR)and Zn^(2+)-assisted DNAzyme cycle with lower cost and simpler procedures.Compared with single DNAzyme amplifier,the proposed TSDR-propelled cascade DNAzyme amplifier exhibited higher sensitivity by releasing more DNAzyme through TSDR to cleave substrate strand during the DNAzyme cycle.Base on this,let-7a could be sensitively detected in the range of 5-50 nmol/L with a detection limit of 64 pmol/L.Furthermore,the dual signal amplification strategy of the cascade DNAzyme amplifier exhibited excellent selectivity to distinguish single-base mismatched DNA strands,which has been successfully applied to the determination of let-7a in blood serum,showing high promise in early cancer diagnosis.展开更多
Detection for deoxyribozyme （DNAzyme） cleavage usually needs complex and time-consuming radial labeling, gel electro- phoresis and autoradiography. A new approach was reported for detection DNAzyme cleavage product ...Detection for deoxyribozyme （DNAzyme） cleavage usually needs complex and time-consuming radial labeling, gel electro- phoresis and autoradiography. A new approach was reported for detection DNAzyme cleavage product based on molecular beacon （MB）. Part of the loop of MB was designed to complementary to DNAzyme cleavage product. MB was employed to monitor ligation process of RNA/DNA complex and to convert directly cleavage product information into fluorescence signal. Detection limit of the assay is 0.02 nmol/L. The cleavage product of 8-17 DNAzyme against HCV-RNA was detected perfectly based on this assay. The method is fast, simple and ultrasensitive, which might hold great promise in DNAzyme reaction and DNAzyme gene therapy.展开更多
As a small catalytic DNA molecule, 10-23 DNAzyme has cleavage ability against complementary RNA. Previous studies of chemical modification have shown that its catalytic core can be further optimized in order to obtain...As a small catalytic DNA molecule, 10-23 DNAzyme has cleavage ability against complementary RNA. Previous studies of chemical modification have shown that its catalytic core can be further optimized in order to obtain more powerful catalytic ability. The analogues of 2＇-deoxyadenosine （5） and 2＇-deoxyguanosine （6） could improve the cleavage ability of the DNAzyme when positioned at positions A9, （32 and G14 in the catalytic core, respectively. Moreover, their combinatorial incorporations were studied, the results implicated that the effect was position-dependent, and positive additive results could be achieved at some positions. The highly conserved G1, G2 and G14 could be optimized by single or combinatorial modification with 2＇-deoxyguanosine analogues. Chemical modifications on the functional groups of the core residues would be a feasible approach for the optimization of 10-23 DNAzyme.展开更多
To overcome ampicillin-resistance of bacteria which is believed to attribute their endogenous B-lactamase, we designed three 10-23 DNAzymes（Dz1, Dz2. Dz3） targeting the coding region of B-lactamase mRNA and examined...To overcome ampicillin-resistance of bacteria which is believed to attribute their endogenous B-lactamase, we designed three 10-23 DNAzymes（Dz1, Dz2. Dz3） targeting the coding region of B-lactamase mRNA and examined their inhibitory capabilities of the ampicillin-resistance of TEM-1 and TEM-3 bacteria. Dz1 was a traditional 10-23 DNAzyme, Dz2 was the mutant of Dz1 by addition of the protected nucleotide to each ann of the enzyme, and Dz3 was a mutant of Dz1 at antisense arms of which phosphorothioate modifications were made. Kinetic analysis, bacterial growth, and β-lactamase activity measurement showed that all the three DNAzymes worked efficiently in vitro and in vivo. A 9 hours bacterial growth inhibition test showed that the inhibition rates of TEM-1 bacteria by Dz1, Dz2, and Dz3 were 27%, 50%, and 29%, respectively. In addition, the inhibition rates of TEM-3 bacteria by those three DNAzymes were found io be 49%, 58%, and 45%, respectively. The current findings suggest that DNAzymes may become potential candidates of alternative inhibitors for bacteria drug-resistance.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 National Talents Program,Double First Class University Plan of Sichuan University,State Key Laboratory of Polymer Materials Engineering(No.sklpme 2020-0301)Natural Science Foundation of Sichuan Province(Nos.2022NSFSC1735,2023NSFSC1097)+5 种基金Fundamental Research Funds for the Central Universities(No.ZYN2022094)National Natural Science Foundation of China(Nos.22178233,22208228)China Postdoctoral Science Foundation(No.2020TQ0209)Fundamental Research Funds for the Central Universities(No.YJ201959)Science and Technology Support Program of Sichuan Province(No.2021YJ0414)Project of Chengdu Science and Technology Bureau(No.2021YF05-02110-SN)。
文摘DNAzyme machines play critical roles in the fields of cell imaging,disease diagnosis,and cancer therapy.However,the applications of DNAzyme machines are limited by the nucleases-induced degradation,non-specific binding of proteins,and insufficient provision of cofactors.Herein,protected DNAzyme machines with different cofactor designs(referred to as Pro Ds)were nanoengineered by the construction of multifunctional metal-phenolic nanoshells to deactivate the interferential proteins,including nucleases and non-specific binding proteins.Moreover,the nanoshells not only facilitate the cellular internalization of Pro Ds but provide specific metal ions acting as cofactors of the designed DNAzymes.Cellular imaging results demonstrated that Pro Ds could effectively and simultaneously monitor multiple tumor-related micro RNAs in living cells.This facile and rapid strategy that encapsulates DNAzyme machines into the protective metal-phenolic nanoshells is anticipated to extend to a wide range of functional nucleic acidsbased biomedical applications.
基金the National Natural Science Foundation of China (Nos. 82172372 and 21904045)COVID-19 Pneumonia Emergency Scientific Research Special Fund of Wuhan (No. EX20D03)the Fundamental Research Funds for the Central Universities (Nos. 2019kfy XJJS169 and 2021yjs CXCY127)。
文摘DNA-functionalized gold nanoparticles are one of the most versatile bionanomaterials for biomedical and clinical diagnosis. Herein, we discovered that the performance of DNAzyme cleaving the substrate is highly related to its length. This intriguing phenomenon only appears at the interfaces of DNAfunctionalized gold nanoparticles. We systematically investigated the causes of this phenomenon. We conjectured that the DNAzyme with extended nucleotides that do not match its substrate strand is vulnerable to non-specific adsorption, electrostatic repulsion, and steric hindrance. Based on our improved understanding of this phenomenon, we have successfully developed a highly sensitive and specific amplifiable biosensor to detect human apurinic/apyrimidinic endonuclease 1.
基金financially supported by the National Natural Science Foundation of China(NSFC,Nos.22074124 and 22134005)the fund of Fundamental Research Funds for the Central Universities(No.XDJK2020TY001)+1 种基金Chongqing Talents Program for Outstanding Scientists(No.cstc2021ycjh-bgzxm0178)the Chongqing Graduate Student Scientific Research Innovation Project(No.CYB21119)。
文摘DNAzyme amplifiers have been extensively explored as a useful sensing platform,but single DNAzyme amplifier is limited in biosensing applications by its low sensitivity.Herein,a cascade DNAzyme amplifier was designed by exploiting concurrent amplification cycle principles of toehold-mediated strand displacement reaction(TSDR)and Zn^(2+)-assisted DNAzyme cycle with lower cost and simpler procedures.Compared with single DNAzyme amplifier,the proposed TSDR-propelled cascade DNAzyme amplifier exhibited higher sensitivity by releasing more DNAzyme through TSDR to cleave substrate strand during the DNAzyme cycle.Base on this,let-7a could be sensitively detected in the range of 5-50 nmol/L with a detection limit of 64 pmol/L.Furthermore,the dual signal amplification strategy of the cascade DNAzyme amplifier exhibited excellent selectivity to distinguish single-base mismatched DNA strands,which has been successfully applied to the determination of let-7a in blood serum,showing high promise in early cancer diagnosis.
基金supported in part by National Basic Research Program of China under Grants(No2009CB421601)Hunan Provincial Science and Technology Program(No2008SK3085)
文摘Detection for deoxyribozyme （DNAzyme） cleavage usually needs complex and time-consuming radial labeling, gel electro- phoresis and autoradiography. A new approach was reported for detection DNAzyme cleavage product based on molecular beacon （MB）. Part of the loop of MB was designed to complementary to DNAzyme cleavage product. MB was employed to monitor ligation process of RNA/DNA complex and to convert directly cleavage product information into fluorescence signal. Detection limit of the assay is 0.02 nmol/L. The cleavage product of 8-17 DNAzyme against HCV-RNA was detected perfectly based on this assay. The method is fast, simple and ultrasensitive, which might hold great promise in DNAzyme reaction and DNAzyme gene therapy.
基金The National Natural Science Foundation of China(Grant No.21572268)
文摘As a small catalytic DNA molecule, 10-23 DNAzyme has cleavage ability against complementary RNA. Previous studies of chemical modification have shown that its catalytic core can be further optimized in order to obtain more powerful catalytic ability. The analogues of 2＇-deoxyadenosine （5） and 2＇-deoxyguanosine （6） could improve the cleavage ability of the DNAzyme when positioned at positions A9, （32 and G14 in the catalytic core, respectively. Moreover, their combinatorial incorporations were studied, the results implicated that the effect was position-dependent, and positive additive results could be achieved at some positions. The highly conserved G1, G2 and G14 could be optimized by single or combinatorial modification with 2＇-deoxyguanosine analogues. Chemical modifications on the functional groups of the core residues would be a feasible approach for the optimization of 10-23 DNAzyme.
文摘目的分析肿瘤远处转移相关蛋白(Ezrin mRNA)的结构,寻找并验证DNAzymes作用的最佳靶点。方法利用RNAstructure与RNAdraw程序分析Ezrin mRNA结构,计算其一、二级结构,两种程序同时计算出碱基未配对的单链成环区,且连续存在4个以上,则将其设为反义技术的靶区域,在此区域内设计DNAzymes的作用靶点,再依据最低自由能原则,运用计算机中的OligoWalk程序进行筛选,以此方式得到各反义技术的作用靶点,以实验方法验证预测结果。结果两种软件预测的共同的单链区共42个,其中完全匹配的单链区21个,编码区具27个。AU1655、AU1751、AU1766、AU1789及GU2623位于连续未配对碱基超过10个的单链区,仅AU1655、AU1751、AU1766、AU1789符合要求。酶切反应结果显示DNAzymes在AU1751位点能够最为理想地切割Ezrin m RNA。结论相对于传统的单纯依靠实验来寻找靶点,核酸二级结构联合热动力学参数能够更精确、快速地处理靶点的设计和选取问题。AU1751位点相对应的DNAzymes不易形成稳定的自身杂合体,有利于DNAzymes结合RNA。
基金the National Natural Science Foundation of China(Nos.20771030 and 20671025)
文摘To overcome ampicillin-resistance of bacteria which is believed to attribute their endogenous B-lactamase, we designed three 10-23 DNAzymes（Dz1, Dz2. Dz3） targeting the coding region of B-lactamase mRNA and examined their inhibitory capabilities of the ampicillin-resistance of TEM-1 and TEM-3 bacteria. Dz1 was a traditional 10-23 DNAzyme, Dz2 was the mutant of Dz1 by addition of the protected nucleotide to each ann of the enzyme, and Dz3 was a mutant of Dz1 at antisense arms of which phosphorothioate modifications were made. Kinetic analysis, bacterial growth, and β-lactamase activity measurement showed that all the three DNAzymes worked efficiently in vitro and in vivo. A 9 hours bacterial growth inhibition test showed that the inhibition rates of TEM-1 bacteria by Dz1, Dz2, and Dz3 were 27%, 50%, and 29%, respectively. In addition, the inhibition rates of TEM-3 bacteria by those three DNAzymes were found io be 49%, 58%, and 45%, respectively. The current findings suggest that DNAzymes may become potential candidates of alternative inhibitors for bacteria drug-resistance.
