Terminal deoxynucleotidyl transferase(Td T) has been characterized as template-independent polymerase using single-stranded DNA(ss DNA) as primers to generate random oligonucleotides. However, the extension performanc...Terminal deoxynucleotidyl transferase(Td T) has been characterized as template-independent polymerase using single-stranded DNA(ss DNA) as primers to generate random oligonucleotides. However, the extension performance of Td T to single-stranded RNA(ss RNA) is vague. By systematically comparing and contrasting the performance of Td T-catalyzed ss DNA and ss RNA extension, it is indicated that the catalytic efficiency of ss RNA as primers was about 3 times lower than ss DNA as primers. Collectively, it is believed that understanding the catalytic performance of Td T will help to design the strategy to synthesize chimeric DNA on 3-OH of ss RNA, which becomes invaluable.展开更多
In this paper, the DNA-templated Ag/Pt bimetallic nanoclusters were successfully synthesized using an optimized synthetic scheme. The obtained DNA-Ag/Pt NCs have an ultrasmall particle size and excellent distribution....In this paper, the DNA-templated Ag/Pt bimetallic nanoclusters were successfully synthesized using an optimized synthetic scheme. The obtained DNA-Ag/Pt NCs have an ultrasmall particle size and excellent distribution. The DNA-Ag/Pt NCs show intrinsic peroxidase-mimicking activity and can effectively catalyze the H2O2-mediated oxidation of a substrate, 3,30,5,50-tetramethylbenzidine（TMB）, to produce a blue colored product. Based on this specific property, we employed the aptamer of VEGF to design a label-free electrochemical biosensor for VEGF detection. Under the optimized experimental conditions, a linear range from 6.0 pmol/L to 20 pmol/L was obtained with a detection limit of 4.6 pmol/L. The proposed biosensor demonstrated its high specificity for VEGF and could directly detect the VEGF concentration in human serum samples of breast cancer patients with satisfactory results. This novel electrochemical aptasensor was simple and convenient to use and was cost-effective and label-free in design, and would hold potential applications in medical diagnosis and treatment.展开更多
Cyanine dyes have attracted more and more interest due to their controllable assembly and disassembly process with biomolecular templates. The self-assembly of cyanine dye not only depend on the environment, but also ...Cyanine dyes have attracted more and more interest due to their controllable assembly and disassembly process with biomolecular templates. The self-assembly of cyanine dye not only depend on the environment, but also on their structures. Here, we report assembly and disassembly of two cyanine dyes,a dimeric cyaine dye(TC-P4) and its corresponding monomer(TC). In PBS, these dyes could form aggregates. The parallel c-myc G-quadruplex as a template causes the transformation of TC-P4 from Haggregates to dimer and monomer; while duplex and single-stranded DNAs could not. The interaction between these DNAs motifs and TC could all induce the appearance of monomer band. Parallel c-myc Gquadruplex could enhance the fluorescence intensity of TC-P4 and TC. The self-assembly and disassembly of TC and TC-P4 could be regulated and used as probes for G-quadruplex recognition from duplex and single-stranded DNAs in solution.展开更多
An ss-DNA gold chip was prepared based on self-assembly of the thiol-derivatized oligonucleotide, and used for the determination of single-stranded binding protein （SSB） by surface plasmon resonance microscopy （SPR...An ss-DNA gold chip was prepared based on self-assembly of the thiol-derivatized oligonucleotide, and used for the determination of single-stranded binding protein （SSB） by surface plasmon resonance microscopy （SPR）. The experiment results showed that SSB binds ss-DNA with high specificity, and relative signal of SPR response is proportional to the concentration of SSB in the range of 0.1-100 ng/mL with a detection limit （S/N = 3） of 0.07 ng/mL.展开更多
DNA-based logic gates promote the development of molecular computing and show enormous potential in the fields of nanotechnology and biotechnology. Dumbbell oligonucleotides(DNA) with poly-thymine(poly-T) loops and a ...DNA-based logic gates promote the development of molecular computing and show enormous potential in the fields of nanotechnology and biotechnology. Dumbbell oligonucleotides(DNA) with poly-thymine(poly-T) loops and a nicked random double strand have been demonstrated to be an efficient template for the formation of fluorescent copper nanoclusters(Cu NCs) in our previous work. Herein, a new platform technology is presented with which to construct molecular logic gates by employing Cu NCs probe as a basic output generator, coupling of functional nucleases as the inputs. Two dumbbell DNAs are used with the difference in stem length(8 bp and 16 bp, respectively). The degradation of DNA templates can be tuned by various nucleic acid enzymes, single-stranded nuclease(S1), double-stranded specific nuclease(DSN), E. coli DNA ligase, exonucleases Ⅰ and Ⅲ. Briefly, S1 can digest both DNA templates, while the cleavage ability of DSN will be resistant by the short stem of SS-DNA(short-stem DNA). Exonuclease Ⅰ and Ⅲ can degrade these two nicked DNA templates, which are inhibited due to the ligation of E. coli DNA ligase. With this novel strategy, a set of logic gates is successfully constructed at the molecular level,including “YES”, “PASS 0”, “OR”, “INHIBIT”, which take the advantages of no label, easy operation, fast speed, high efficiency and low cost. Furthermore, S1 nuclease, as the biomarker of numerous carcinogens,is selectively detected in the range of 0.05–50 U/m L with the detection limit of 0.005 U/m L(1×10^(−6)U)based on this platform.展开更多
According to ultraviolet （UV）-vis absorption spectra recorded in the DNA metallization process, DNA-templated Co/Cu binary nanoparticle chains are fabricated by incubating genome DNA of paralichthys olivaceus muscle...According to ultraviolet （UV）-vis absorption spectra recorded in the DNA metallization process, DNA-templated Co/Cu binary nanoparticle chains are fabricated by incubating genome DNA of paralichthys olivaceus muscle in CoCl2 and CuCl2 mixture solution for 20 hours and reducing the complex for 2 hours. Transmission electron microscopy observation indicates that Co and Cu nanoparticles with 20 nm in diameter were randomly dispersed on the DNA template. The superconducting quantum interference device （SQUID） measurements display that the magnetic interaction between cobalt particles is greatly decreased by the copper particle. With increasing copper content, the coercivity of the systems enhance from 9 Oe to 100 Oe （1 Oe=79.5775 A/m）.展开更多
[Objective] This study aimed to optimize the PCR amplification conditions for random ssDNA pool in SELEX technology. [Method] L16（45） orthogonal experimental design was adopted for optimization of five important fac...[Objective] This study aimed to optimize the PCR amplification conditions for random ssDNA pool in SELEX technology. [Method] L16（45） orthogonal experimental design was adopted for optimization of five important factors affecting PCR reaction system for random single-stranded DNA pool including Mg2＋ concentration, dNTP concentration, amount of Taq DNA polymerase, primer concentration and amount of random single-stranded DNA pool at four levels. Meanwhile, the annealing temperature and number of PCR reaction cycles were optimized to establish the optimal reaction system and PCR procedure. [Result] The optimal combination of PCR reaction system for random ssDNA pool was obtained, with a total system volume of 20 μl containing 2.0 μl of 10 × Buffer, 0.5 ng of random ssDNA pool, 2.5 mmol/L Mg2＋, 0.25 mmol/L dNTP Mixture, 0.6 μmol/L upstream and downstream primers and 1.5 U of Taq DNA polymerase; the optimal annealing temperature was 68 ℃ and the optimal number of cycles was 12. Under the above conditions, clear and stable bands with high specificity for random ssDNA pool were amplified. [Conclusion] This study laid the foundation for selection of parameters with higher specificity in SELEX technology.展开更多
Single-strand breaks （SSBs） can occur in cells either directly, or indirectly following initiation of base excision repair （BER）. SSBs generally have blocked termini lacking the conventional 5＇-phosphate and 3＇-...Single-strand breaks （SSBs） can occur in cells either directly, or indirectly following initiation of base excision repair （BER）. SSBs generally have blocked termini lacking the conventional 5＇-phosphate and 3＇-hydroxyl groups and require further processing prior to DNA synthesis and ligation. XRCC1 is devoid of any known enzymatic activity, but it can physically interact with other proteins involved in all stages of the overlapping SSB repair and BER pathways, including those that conduct the rate-limiting end-tailoring, and in many cases can stimulate their enzymatic activities. XRCC1^-/- mouse fibroblasts are most hypersensitive to agents that produce DNA lesions repaired by monofunctional glycosylase-initiated BER and that result in formation of indirect SSBs. A requirement for the deoxyribose phosphate lyase activity of DNA polymerase β （pol β） is specific to this pathway, whereas pol β is implicated in gap-filling during repair of many types of SSBs. Elevated levels of strand breaks, and diminished repair, have been demonstrated in MMS- treated XRCC1^-/-, and to a lesser extent in pol β^-/- cell lines, compared with wild-type cells. Thus a strong correlation is observed between cellular sensitivity to MMS and the ability of cells to repair MMS-induced damage. Exposure of wild-type and polβ^-/- cells to an inhibitor of PARP activity dramatically potentiates MMS-induced cytotoxicity. XRCC1^-/- cells are also sensitized by PARP inhibition demonstrating that PARP-mediated poly（ADP-ribosyl）ation plays a role in modulation of cytotoxicity beyond recruitment of XRCC 1 to sites of DNA damage.展开更多
目的建立新型冠状病毒mRNA疫苗模板DNA残留数字微滴PCR(droplet digital PCR,ddPCR)检测方法并进行验证,为新型冠状病毒mRNA疫苗的质量控制提供技术支持。方法以不同质粒共有DNA序列复制子为靶基因,设计特异性引物和探针,建立新型冠状病...目的建立新型冠状病毒mRNA疫苗模板DNA残留数字微滴PCR(droplet digital PCR,ddPCR)检测方法并进行验证,为新型冠状病毒mRNA疫苗的质量控制提供技术支持。方法以不同质粒共有DNA序列复制子为靶基因,设计特异性引物和探针,建立新型冠状病毒mRNA疫苗模板DNA残留ddPCR检测方法,并进行线性范围、重复性、准确度、中间精密度、耐用性、检测限、定量限、专属性验证;用建立的方法检测不同企业、不同变异株新型冠状病毒mRNA疫苗模板DNA残留。结果选择58℃为建立的ddPCR法的最优退火温度。检测拷贝数在19~4729 copies/μL范围内,线性良好,R^(2)=0.9999;重复性变异系数(coefficient of variation,CV)<10%;准确度回收率在99%121%之间,CV<10%;中间精密度和耐用性CV均<10%;建立的方法检测限为9 copies/μL,定量限为26 copies/μL,专属性良好。4家不同企业、不同变异株新型冠状病毒mRNA疫苗模板DNA残留CV均≤15%,回收率在79%138%之间。结论建立的新型冠状病毒mRNA疫苗模板DNA残留ddPCR检测方法灵敏度高、稳定性好、专属性强、准确度高,适用于新型冠状病毒mRNA疫苗模板DNA残留量检测。展开更多
Monolayer of octadecylamine（ODA） and salmon sperm DNA or salmon sperm DNA-Cd complex were studied on air-water interface by π-A isotherm. After being co-transferred onto substrates by Langmuir-Blodgett technique, a...Monolayer of octadecylamine（ODA） and salmon sperm DNA or salmon sperm DNA-Cd complex were studied on air-water interface by π-A isotherm. After being co-transferred onto substrates by Langmuir-Blodgett technique, atomic force microscope（AFM） measurements show the DNA molecules are packed into lines in the film, due to the interactions between the ODA and DNA molecules. By exposing the ODA and DNA-Cd complex LB film to H2S, needle-like CdS nanoparticles were formed along the DNA lines as characterized by transmission electron microscope（TEM）. Electron diffraction（ED） image indicates that the so prepared needle-like CdS is a new kind of nanostructured materials.展开更多
基金supported by the National Natural Science Foundation of China (NSFC, Nos. 21927814 and 21772143 to J.Y. Zhang)the National Science Foundation of Tianjin (Nos. 20YDTPJC00090, 19ZXDBSY00070 and 20YFZCSY00990 to X.Q. Gong)。
文摘Terminal deoxynucleotidyl transferase(Td T) has been characterized as template-independent polymerase using single-stranded DNA(ss DNA) as primers to generate random oligonucleotides. However, the extension performance of Td T to single-stranded RNA(ss RNA) is vague. By systematically comparing and contrasting the performance of Td T-catalyzed ss DNA and ss RNA extension, it is indicated that the catalytic efficiency of ss RNA as primers was about 3 times lower than ss DNA as primers. Collectively, it is believed that understanding the catalytic performance of Td T will help to design the strategy to synthesize chimeric DNA on 3-OH of ss RNA, which becomes invaluable.
基金support of the National Natural Science Foundation of China (Nos. 21375017, 21105012 and 21205015)the National Science Foundation for Distinguished Young Scholars of Fujian Province (No. 2013J06003)+3 种基金the Key Project of Fujian Science and Technology (No. 2013Y0045)the Program for New Century Excellent Talents of Colleges and Universities in Fujian Province (Nos. JA13130 and JA13088)the Program for Fujian University Outstanding Youth Scientific Research (Nos. JA11105 and JA10295)the Foundation of Fuzhou Science and Technology Bureau (No. 2013-S-122-4)
文摘In this paper, the DNA-templated Ag/Pt bimetallic nanoclusters were successfully synthesized using an optimized synthetic scheme. The obtained DNA-Ag/Pt NCs have an ultrasmall particle size and excellent distribution. The DNA-Ag/Pt NCs show intrinsic peroxidase-mimicking activity and can effectively catalyze the H2O2-mediated oxidation of a substrate, 3,30,5,50-tetramethylbenzidine（TMB）, to produce a blue colored product. Based on this specific property, we employed the aptamer of VEGF to design a label-free electrochemical biosensor for VEGF detection. Under the optimized experimental conditions, a linear range from 6.0 pmol/L to 20 pmol/L was obtained with a detection limit of 4.6 pmol/L. The proposed biosensor demonstrated its high specificity for VEGF and could directly detect the VEGF concentration in human serum samples of breast cancer patients with satisfactory results. This novel electrochemical aptasensor was simple and convenient to use and was cost-effective and label-free in design, and would hold potential applications in medical diagnosis and treatment.
基金financially supported by General Program of the National Natural Science Foundation of China(Nos. 21472197. 21675126 and 21778058)"Science and Technology Service Network Initiative" of the Chinese Academy of Sciences
文摘Cyanine dyes have attracted more and more interest due to their controllable assembly and disassembly process with biomolecular templates. The self-assembly of cyanine dye not only depend on the environment, but also on their structures. Here, we report assembly and disassembly of two cyanine dyes,a dimeric cyaine dye(TC-P4) and its corresponding monomer(TC). In PBS, these dyes could form aggregates. The parallel c-myc G-quadruplex as a template causes the transformation of TC-P4 from Haggregates to dimer and monomer; while duplex and single-stranded DNAs could not. The interaction between these DNAs motifs and TC could all induce the appearance of monomer band. Parallel c-myc Gquadruplex could enhance the fluorescence intensity of TC-P4 and TC. The self-assembly and disassembly of TC and TC-P4 could be regulated and used as probes for G-quadruplex recognition from duplex and single-stranded DNAs in solution.
基金the Science Foundation of the National Education Ministry （No, 206096） the Education Department of Hubei Province （No. Z200522002）.
文摘An ss-DNA gold chip was prepared based on self-assembly of the thiol-derivatized oligonucleotide, and used for the determination of single-stranded binding protein （SSB） by surface plasmon resonance microscopy （SPR）. The experiment results showed that SSB binds ss-DNA with high specificity, and relative signal of SPR response is proportional to the concentration of SSB in the range of 0.1-100 ng/mL with a detection limit （S/N = 3） of 0.07 ng/mL.
基金the projects of Innovative research team of high-level local universities in Shanghai and a key laboratory program of the Education Commission of Shanghai Municipality (No. ZDSYS14005)Program for high-level local universities in Shanghai (No. IDF301027/022)+1 种基金Shanghai Agriculture Science and Technology Support Project (No. 21N31900500)the National Natural Science Foundation of China (No. 21505023)
文摘DNA-based logic gates promote the development of molecular computing and show enormous potential in the fields of nanotechnology and biotechnology. Dumbbell oligonucleotides(DNA) with poly-thymine(poly-T) loops and a nicked random double strand have been demonstrated to be an efficient template for the formation of fluorescent copper nanoclusters(Cu NCs) in our previous work. Herein, a new platform technology is presented with which to construct molecular logic gates by employing Cu NCs probe as a basic output generator, coupling of functional nucleases as the inputs. Two dumbbell DNAs are used with the difference in stem length(8 bp and 16 bp, respectively). The degradation of DNA templates can be tuned by various nucleic acid enzymes, single-stranded nuclease(S1), double-stranded specific nuclease(DSN), E. coli DNA ligase, exonucleases Ⅰ and Ⅲ. Briefly, S1 can digest both DNA templates, while the cleavage ability of DSN will be resistant by the short stem of SS-DNA(short-stem DNA). Exonuclease Ⅰ and Ⅲ can degrade these two nicked DNA templates, which are inhibited due to the ligation of E. coli DNA ligase. With this novel strategy, a set of logic gates is successfully constructed at the molecular level,including “YES”, “PASS 0”, “OR”, “INHIBIT”, which take the advantages of no label, easy operation, fast speed, high efficiency and low cost. Furthermore, S1 nuclease, as the biomarker of numerous carcinogens,is selectively detected in the range of 0.05–50 U/m L with the detection limit of 0.005 U/m L(1×10^(−6)U)based on this platform.
基金Project partially supported by the Key Project of the Ministry of Education of China(Grant No.109025)
文摘According to ultraviolet （UV）-vis absorption spectra recorded in the DNA metallization process, DNA-templated Co/Cu binary nanoparticle chains are fabricated by incubating genome DNA of paralichthys olivaceus muscle in CoCl2 and CuCl2 mixture solution for 20 hours and reducing the complex for 2 hours. Transmission electron microscopy observation indicates that Co and Cu nanoparticles with 20 nm in diameter were randomly dispersed on the DNA template. The superconducting quantum interference device （SQUID） measurements display that the magnetic interaction between cobalt particles is greatly decreased by the copper particle. With increasing copper content, the coercivity of the systems enhance from 9 Oe to 100 Oe （1 Oe=79.5775 A/m）.
基金Supported by Central University Basic Research Operating Expenses Special Fund(XDJK2011C026)Southwest University Doctoral Fund(09BSR04)~~
文摘[Objective] This study aimed to optimize the PCR amplification conditions for random ssDNA pool in SELEX technology. [Method] L16（45） orthogonal experimental design was adopted for optimization of five important factors affecting PCR reaction system for random single-stranded DNA pool including Mg2＋ concentration, dNTP concentration, amount of Taq DNA polymerase, primer concentration and amount of random single-stranded DNA pool at four levels. Meanwhile, the annealing temperature and number of PCR reaction cycles were optimized to establish the optimal reaction system and PCR procedure. [Result] The optimal combination of PCR reaction system for random ssDNA pool was obtained, with a total system volume of 20 μl containing 2.0 μl of 10 × Buffer, 0.5 ng of random ssDNA pool, 2.5 mmol/L Mg2＋, 0.25 mmol/L dNTP Mixture, 0.6 μmol/L upstream and downstream primers and 1.5 U of Taq DNA polymerase; the optimal annealing temperature was 68 ℃ and the optimal number of cycles was 12. Under the above conditions, clear and stable bands with high specificity for random ssDNA pool were amplified. [Conclusion] This study laid the foundation for selection of parameters with higher specificity in SELEX technology.
文摘Single-strand breaks （SSBs） can occur in cells either directly, or indirectly following initiation of base excision repair （BER）. SSBs generally have blocked termini lacking the conventional 5＇-phosphate and 3＇-hydroxyl groups and require further processing prior to DNA synthesis and ligation. XRCC1 is devoid of any known enzymatic activity, but it can physically interact with other proteins involved in all stages of the overlapping SSB repair and BER pathways, including those that conduct the rate-limiting end-tailoring, and in many cases can stimulate their enzymatic activities. XRCC1^-/- mouse fibroblasts are most hypersensitive to agents that produce DNA lesions repaired by monofunctional glycosylase-initiated BER and that result in formation of indirect SSBs. A requirement for the deoxyribose phosphate lyase activity of DNA polymerase β （pol β） is specific to this pathway, whereas pol β is implicated in gap-filling during repair of many types of SSBs. Elevated levels of strand breaks, and diminished repair, have been demonstrated in MMS- treated XRCC1^-/-, and to a lesser extent in pol β^-/- cell lines, compared with wild-type cells. Thus a strong correlation is observed between cellular sensitivity to MMS and the ability of cells to repair MMS-induced damage. Exposure of wild-type and polβ^-/- cells to an inhibitor of PARP activity dramatically potentiates MMS-induced cytotoxicity. XRCC1^-/- cells are also sensitized by PARP inhibition demonstrating that PARP-mediated poly（ADP-ribosyl）ation plays a role in modulation of cytotoxicity beyond recruitment of XRCC 1 to sites of DNA damage.
文摘目的建立新型冠状病毒mRNA疫苗模板DNA残留数字微滴PCR(droplet digital PCR,ddPCR)检测方法并进行验证,为新型冠状病毒mRNA疫苗的质量控制提供技术支持。方法以不同质粒共有DNA序列复制子为靶基因,设计特异性引物和探针,建立新型冠状病毒mRNA疫苗模板DNA残留ddPCR检测方法,并进行线性范围、重复性、准确度、中间精密度、耐用性、检测限、定量限、专属性验证;用建立的方法检测不同企业、不同变异株新型冠状病毒mRNA疫苗模板DNA残留。结果选择58℃为建立的ddPCR法的最优退火温度。检测拷贝数在19~4729 copies/μL范围内,线性良好,R^(2)=0.9999;重复性变异系数(coefficient of variation,CV)<10%;准确度回收率在99%121%之间,CV<10%;中间精密度和耐用性CV均<10%;建立的方法检测限为9 copies/μL,定量限为26 copies/μL,专属性良好。4家不同企业、不同变异株新型冠状病毒mRNA疫苗模板DNA残留CV均≤15%,回收率在79%138%之间。结论建立的新型冠状病毒mRNA疫苗模板DNA残留ddPCR检测方法灵敏度高、稳定性好、专属性强、准确度高,适用于新型冠状病毒mRNA疫苗模板DNA残留量检测。
文摘Monolayer of octadecylamine（ODA） and salmon sperm DNA or salmon sperm DNA-Cd complex were studied on air-water interface by π-A isotherm. After being co-transferred onto substrates by Langmuir-Blodgett technique, atomic force microscope（AFM） measurements show the DNA molecules are packed into lines in the film, due to the interactions between the ODA and DNA molecules. By exposing the ODA and DNA-Cd complex LB film to H2S, needle-like CdS nanoparticles were formed along the DNA lines as characterized by transmission electron microscope（TEM）. Electron diffraction（ED） image indicates that the so prepared needle-like CdS is a new kind of nanostructured materials.
