Developing nanomaterial-based enzyme mimics for DNA cleavage is an interesting challenge and it has many potential applications.Single-layered graphene oxide(GO)is an excellent platform for DNA adsorption.In addition,...Developing nanomaterial-based enzyme mimics for DNA cleavage is an interesting challenge and it has many potential applications.Single-layered graphene oxide(GO)is an excellent platform for DNA adsorption.In addition,GO has been employed for photosensitized generation of reactive oxygen species(ROS).Herein,we demonstrate that GO sheets could cleave DNA as anuclease mimicking nanozyme in the presence of UV or blue light.For various DNA sequences and lengths,well-defined product bands were observed along with photobleaching of the fluorophore label on the DNA.Different from previously reported GO cleavage of DNA,our method did not require metal ions such as Cu^2+.Fluorescence spectroscopy suggested a high adsorption affinity between GO and DNA.For comparison,although zero-dimensional fluorescent carbon dots(C-dots)had higher photosensitivity ir terms of producing ROS,their cleavage activity was much lower and only smeared cleavage products were observed,indicatingthat the ROS acted on the DNA in solution.Based on the results,GO behaved like a classic heterogeneous catalyst following substrate adsorption,reaction,and product desorption steps.This simple strategy may help in the design of new nanozymes by introducing light.展开更多
A copper-bispyridylpyrrolide complex [Cu(PDPH)Cl](PDPH = 2,5-bis(2′-pyridyl)pyrrole) was synthesized and characterized. The complex crystallizes in the orthorhombic system with space group Pccn, a = 0.9016(3) nm, b =...A copper-bispyridylpyrrolide complex [Cu(PDPH)Cl](PDPH = 2,5-bis(2′-pyridyl)pyrrole) was synthesized and characterized. The complex crystallizes in the orthorhombic system with space group Pccn, a = 0.9016(3) nm, b = 1.0931(4) nm, c =2.5319(8) nm, and V = 2.4951(15) nm3. The copper center is situated in a square planar geometry. The interaction of the copper(II)complex with calf thymus DNA(CT-DNA) was investigated by electronic absorption, circular dichroism(CD) and fluorescence spectra. It is proposed that the complex binds to CT-DNA through groove binding mode. Nuclease activity of the complex was also studied by gel electrophoresis method. The complex can efficiently cleave supercoiled p BR322 DNA in the presence of ascorbate(H2A) via oxidative pathway. The preliminary mechanism of DNA cleavage by the complex with different inhibiting reagents indicates that the hydroxyl radicals were involved as the active species in the DNA cleavage process.展开更多
New metal-free DNA cleaving reagent 1,1,4,7-triazacrown(TACN)both with aminoethyl,hydroxyethyl side arms and a planar anthraquinone linked by an alkyl(1,6-hexamethylene)spacer has been synthesized and characterized by...New metal-free DNA cleaving reagent 1,1,4,7-triazacrown(TACN)both with aminoethyl,hydroxyethyl side arms and a planar anthraquinone linked by an alkyl(1,6-hexamethylene)spacer has been synthesized and characterized by NMR and MS spectrometry.For comparison,the corresponding aminoethyl,hydroxyethyl triazacrown derivative 2 without the anthraquinone has also been synthesized.DNA-binding properties via fluorescence and CD spectroscopy indicate that the binding affinity of 1 with DNA is much stronger than that of 2.Agarose gel electrophoresis was used to assess plasmid pUC19 DNA cleavage.Kinetic data of DNA cleavage promoted by 1,2 and parent triazacrown(TACN)3 under physiological condition give the 15-fold and 234-fold rate acceleration of compound 1 over 2 and parent triazacrown 3.Radical scavenger inhibition study suggests that DNA cleavage promoted by 1 may be a non-oxidative pathway through the transphosphorylation and then hydrolysis.The dramatic rate acceleration is due not only to the anthraquinone moiety of compound 1 intercalating into DNA base pairs via stacking interaction,but also the cooperative catalysis of the nucleophilic hydroxyl and the electrophilic ammonium group for the cleavage of phosphodiester of DNA.展开更多
The cleavage reactions of Ser-His and its N-terminal phosphorylated form - N-(O.O-diisopropyl) phosphoryl seryl-histidine (DIPP-Ser-His) were studied on DNA. It was found that the phosporylation of Ser-His caused the ...The cleavage reactions of Ser-His and its N-terminal phosphorylated form - N-(O.O-diisopropyl) phosphoryl seryl-histidine (DIPP-Ser-His) were studied on DNA. It was found that the phosporylation of Ser-His caused the lost of the cleavage activity on DNA. The result might give some clue on the regulation of the activity of protein by展开更多
MicroRNAs(miRNAs),especially exosomal miRNAs,are promising noninvasive biomarkers in early-stage cancer diagnosis and disease treatment monitoring.However,their precise and sensitive quantification remains challenging...MicroRNAs(miRNAs),especially exosomal miRNAs,are promising noninvasive biomarkers in early-stage cancer diagnosis and disease treatment monitoring.However,their precise and sensitive quantification remains challenging due to their small size and low abundance.Herein,we have developed a nanoparticle-confined DNA walker strategy for the specific detection of miRNA.In the existence of the target miRNA;the on-particle DNA walking reaction will be initiated,providing a fluorescence-positive nanoparticle.Otherwise,the nanoparticle would be fluorescence-negative.Utilizing the total internal reflection fluorescent microscope(TIRFM)to digitally count the fluorescence-positive nanoparticles,the proposed method possesses a detection limit of 0.2 pmol/L miRNA and can accurately distinguish the single-base mismatched target.This design combines the merits of the DNA walker for signal amplification and the TIRFM for highly sensitive detection,paving a new way for the digital counting-based analysis of exosomal miRNAs.展开更多
The control of protein functions with light is valuable for spatiotemporal probing of biological systems.Current small-molecule photo-modulation methods include the light-induced uncaging of inhibitors and chromophore...The control of protein functions with light is valuable for spatiotemporal probing of biological systems.Current small-molecule photo-modulation methods include the light-induced uncaging of inhibitors and chromophore-assisted light inactivation with reactive oxygen species(ROS).However,the constant target protein expression results in inadequate photo-modulation efficiency,particularly for less potent inhibitors and chromophores.Herein,we report a novel bifunctional small-molecule ligands strategy to photo-modulate gene-editing enzymes CRISPR/Cas9.A coumarin-derived small-molecule ligand Bhc-BRD0539 is developed to uncage the active inhibitor upon light irradiation and to generate ROS in the Cas9 proximity for the dual inhibition of Cas9 activity.Our results highlight the synergistic photo-modulation with bifunctional small-molecule ligands,which offers a valuable addition to current CRISPR/Cas9 photo-modulation technologies and may extend to other protein classes.展开更多
基金Funding for this work was from the Natural Sciences and Engineering Research Council of Canada(NSERC)the National Natural Science Foundation of China(No.U19A2005).
文摘Developing nanomaterial-based enzyme mimics for DNA cleavage is an interesting challenge and it has many potential applications.Single-layered graphene oxide(GO)is an excellent platform for DNA adsorption.In addition,GO has been employed for photosensitized generation of reactive oxygen species(ROS).Herein,we demonstrate that GO sheets could cleave DNA as anuclease mimicking nanozyme in the presence of UV or blue light.For various DNA sequences and lengths,well-defined product bands were observed along with photobleaching of the fluorophore label on the DNA.Different from previously reported GO cleavage of DNA,our method did not require metal ions such as Cu^2+.Fluorescence spectroscopy suggested a high adsorption affinity between GO and DNA.For comparison,although zero-dimensional fluorescent carbon dots(C-dots)had higher photosensitivity ir terms of producing ROS,their cleavage activity was much lower and only smeared cleavage products were observed,indicatingthat the ROS acted on the DNA in solution.Based on the results,GO behaved like a classic heterogeneous catalyst following substrate adsorption,reaction,and product desorption steps.This simple strategy may help in the design of new nanozymes by introducing light.
基金Project(21001118)supported by National Natural Science Foundation of ChinaProject(12JJ3016)supported by Natural Science Foundation of Hunan Province,China
文摘A copper-bispyridylpyrrolide complex [Cu(PDPH)Cl](PDPH = 2,5-bis(2′-pyridyl)pyrrole) was synthesized and characterized. The complex crystallizes in the orthorhombic system with space group Pccn, a = 0.9016(3) nm, b = 1.0931(4) nm, c =2.5319(8) nm, and V = 2.4951(15) nm3. The copper center is situated in a square planar geometry. The interaction of the copper(II)complex with calf thymus DNA(CT-DNA) was investigated by electronic absorption, circular dichroism(CD) and fluorescence spectra. It is proposed that the complex binds to CT-DNA through groove binding mode. Nuclease activity of the complex was also studied by gel electrophoresis method. The complex can efficiently cleave supercoiled p BR322 DNA in the presence of ascorbate(H2A) via oxidative pathway. The preliminary mechanism of DNA cleavage by the complex with different inhibiting reagents indicates that the hydroxyl radicals were involved as the active species in the DNA cleavage process.
基金This work was supported by the National Science Foundation(No.31170536)the Foundation of Priority Academic Program Development(PAPD)of Jiangsu Higher Education Institutions(2010).
文摘New metal-free DNA cleaving reagent 1,1,4,7-triazacrown(TACN)both with aminoethyl,hydroxyethyl side arms and a planar anthraquinone linked by an alkyl(1,6-hexamethylene)spacer has been synthesized and characterized by NMR and MS spectrometry.For comparison,the corresponding aminoethyl,hydroxyethyl triazacrown derivative 2 without the anthraquinone has also been synthesized.DNA-binding properties via fluorescence and CD spectroscopy indicate that the binding affinity of 1 with DNA is much stronger than that of 2.Agarose gel electrophoresis was used to assess plasmid pUC19 DNA cleavage.Kinetic data of DNA cleavage promoted by 1,2 and parent triazacrown(TACN)3 under physiological condition give the 15-fold and 234-fold rate acceleration of compound 1 over 2 and parent triazacrown 3.Radical scavenger inhibition study suggests that DNA cleavage promoted by 1 may be a non-oxidative pathway through the transphosphorylation and then hydrolysis.The dramatic rate acceleration is due not only to the anthraquinone moiety of compound 1 intercalating into DNA base pairs via stacking interaction,but also the cooperative catalysis of the nucleophilic hydroxyl and the electrophilic ammonium group for the cleavage of phosphodiester of DNA.
文摘The cleavage reactions of Ser-His and its N-terminal phosphorylated form - N-(O.O-diisopropyl) phosphoryl seryl-histidine (DIPP-Ser-His) were studied on DNA. It was found that the phosporylation of Ser-His caused the lost of the cleavage activity on DNA. The result might give some clue on the regulation of the activity of protein by
基金the National Natural Science Foundation of China(Nos.22074088 and 21904083)the Program for Changjiang Scholars and Innovative Research Team in University(IRT_15R43)the Fundamental Research Funds for the Central Universities(Nos.GK202003038,GK201901003,2020TS089,GK202101001).
文摘MicroRNAs(miRNAs),especially exosomal miRNAs,are promising noninvasive biomarkers in early-stage cancer diagnosis and disease treatment monitoring.However,their precise and sensitive quantification remains challenging due to their small size and low abundance.Herein,we have developed a nanoparticle-confined DNA walker strategy for the specific detection of miRNA.In the existence of the target miRNA;the on-particle DNA walking reaction will be initiated,providing a fluorescence-positive nanoparticle.Otherwise,the nanoparticle would be fluorescence-negative.Utilizing the total internal reflection fluorescent microscope(TIRFM)to digitally count the fluorescence-positive nanoparticles,the proposed method possesses a detection limit of 0.2 pmol/L miRNA and can accurately distinguish the single-base mismatched target.This design combines the merits of the DNA walker for signal amplification and the TIRFM for highly sensitive detection,paving a new way for the digital counting-based analysis of exosomal miRNAs.
基金supported by the National Natural Science Foundation of China(22337005,22277133,91753126)the Youth Innovation Promotion Association(CAS 2023266)+1 种基金the CAS Interdisciplinary Innovation Team(JCTD-2020-16)the Program of Shanghai Academic/Technology Research Leader(21XD1424700).
文摘The control of protein functions with light is valuable for spatiotemporal probing of biological systems.Current small-molecule photo-modulation methods include the light-induced uncaging of inhibitors and chromophore-assisted light inactivation with reactive oxygen species(ROS).However,the constant target protein expression results in inadequate photo-modulation efficiency,particularly for less potent inhibitors and chromophores.Herein,we report a novel bifunctional small-molecule ligands strategy to photo-modulate gene-editing enzymes CRISPR/Cas9.A coumarin-derived small-molecule ligand Bhc-BRD0539 is developed to uncage the active inhibitor upon light irradiation and to generate ROS in the Cas9 proximity for the dual inhibition of Cas9 activity.Our results highlight the synergistic photo-modulation with bifunctional small-molecule ligands,which offers a valuable addition to current CRISPR/Cas9 photo-modulation technologies and may extend to other protein classes.
