Protein self-labeling tags achieve selective fusion and labeling of target proteins through genetic coding technology,but require exogenous fluorescent probes with fluorogenicity for protein tag binding to have the pe...Protein self-labeling tags achieve selective fusion and labeling of target proteins through genetic coding technology,but require exogenous fluorescent probes with fluorogenicity for protein tag binding to have the performance of wash-free fluorescence imaging in live cells.In this paper,we reported a fluorogenic probe 1 capable of ratiometric fluorescence recognition of SNAP-tag proteins.In this probe,the O6-benzylguanine derivative of 3-hydroxy-1,8-naphthalimide underwent a selective covalent linkage reaction with SNAP-tag protein.The hydroxyl group on the naphthalimide fluorophore formed a hydrogen bond with the functional group near the protein cavity.The excited state proton transfer occurred after illumination,to obtain the ratio fluorescence signal from blue emission to red emission,realizing the wash-free fluorescence imaging of the target proteins.展开更多
The combination of super-resolution microscopy and synthetic fluorescence probes has emerged as a universal tool to monitor dynamic biological events at the nanometer scale.However,the limited site-specificity and flu...The combination of super-resolution microscopy and synthetic fluorescence probes has emerged as a universal tool to monitor dynamic biological events at the nanometer scale.However,the limited site-specificity and fluorogenicity of synthetic fluorescent probes make it still difficult to realize long-term super-resolution imaging.Herein,we introduce a dynamic aggregation mediated SNAP-tag fluorogenic probe,BGAN-Aze,which can specifically bind to various SNAP-tag fusion proteins with 41-fold fluorescence enhancement.The equilibrium between the non-fluorescent aggregate/dimer(A–D)and the fluorescent monomer(M)of BGAN-Aze acts as an effective method to reduce the fluorescence background and endow BGAN-Aze with the capability of conducting washing-free super-resolution imaging of various intracellular and extracellular proteins.Using this probe,we monitored multiple dynamic biological events,such as MMC,mitophagy,the fusion of nucleolus,and the growth and contact of filopodia.We expect that BGAN-Aze will become a widely used SNAP-tag for super-resolution imaging of dynamic biological events and the A-D-M equilibrium can be a general strategy for designing fluorogenic probes.展开更多
Protein labeling by using a protein tag and tag-specific fluorescent probes is increasingly becoming a useful technique for the real-time imaging of proteins in living cells. SNAP-tag as one of the most prominent fusi...Protein labeling by using a protein tag and tag-specific fluorescent probes is increasingly becoming a useful technique for the real-time imaging of proteins in living cells. SNAP-tag as one of the most prominent fusion tags has been widely used and already commercially available. Recently, various fluorogenic probes for SNAP-tag based protein labeling were reported. Owing to turn-on fluorescence response, fluorogenic probes for SNAP-tag minimize the fluorescence background caused by unreacted or nonspecifically bound probes and allow for direct imaging in living cells without wash-out steps. Thus,real-time analysis of protein localization, dynamics and interactions has been made possible by SNAP-tag fluorogenic probes. In this review,we describe the design strategies of fluorogenic probes for SNAP-tag and their applications in cellular protein labeling.展开更多
Monitoring dynamics of mitochondria has become an essential approach to explore the function of mitochondria in living cells with the emergence of super-resolution fluorescence microscopy.However,long-term super-resol...Monitoring dynamics of mitochondria has become an essential approach to explore the function of mitochondria in living cells with the emergence of super-resolution fluorescence microscopy.However,long-term super-resolution imaging of mitochondria is still challenging due to the lack of photostable fluorescent probes and stable mitochondria-specific markers which are not affected by the changes of mitochondrial membrane potential.Here,we introduce a method for long-term imaging mitochondrial dynamic through the SNAP-tag fluorogenic probe based on 4-azetidinyl-naphthalimide derivatives.Using structured illumination microscopy(SIM),we observed the fusion and fission of mitochondria over a course of 16 min at 109 nm resolution.Furthermore,the interactions as well as fusion between mitochondria and lysosomes were studied during mitophagy at the nanoscale.Convincingly,the combination of SNAP-tag fluorogenic probes and super-resolution fluorescence microscopy will offer a new way to monitor dynamic mitochondria in living cells.展开更多
Methyl-CpG-binding domain(MBD)proteins can specifically recognize and bind methylated CpG sites of DNA,thus repress gene transcription.In this study,we designed and expressed two recombinant proteins,MBD2b and SNAP-MB...Methyl-CpG-binding domain(MBD)proteins can specifically recognize and bind methylated CpG sites of DNA,thus repress gene transcription.In this study,we designed and expressed two recombinant proteins,MBD2b and SNAP-MBD2b,in E.coli.An optimized protocol was developed to purify the proteins using Ni-NTA affinity cartridge and cation exchange resin.The engineered proteins purified by this method exhibited more than 93%purity and high binding avidity.We found that both SNAP-MBD2b and MBD2b were prone to aggregate during dialysis.However,this could be prevented by the use of 0.3 mol/L NaCl.The fusion of SNAP-tag with MBD2b significantly enhanced the expression of MBD2b protein in E.coli and reduced the adsorption of MBD2b on solid interfaces involved in protein purification and immobilization.The engineered proteins can be used for the study of interaction with methylated DNA and the assays for DNA methylation.展开更多
基金supported by the National Natural Science Foundation of China(Nos.22225806,22078314 and 22278394)Dalian Institute of Chemical Physics(Nos.DICPI202227 and DICPI202142)。
文摘Protein self-labeling tags achieve selective fusion and labeling of target proteins through genetic coding technology,but require exogenous fluorescent probes with fluorogenicity for protein tag binding to have the performance of wash-free fluorescence imaging in live cells.In this paper,we reported a fluorogenic probe 1 capable of ratiometric fluorescence recognition of SNAP-tag proteins.In this probe,the O6-benzylguanine derivative of 3-hydroxy-1,8-naphthalimide underwent a selective covalent linkage reaction with SNAP-tag protein.The hydroxyl group on the naphthalimide fluorophore formed a hydrogen bond with the functional group near the protein cavity.The excited state proton transfer occurred after illumination,to obtain the ratio fluorescence signal from blue emission to red emission,realizing the wash-free fluorescence imaging of the target proteins.
基金National Natural Science Foundation of China,Grant/Award Numbers:22078314,21878286,21908216Dalian Institute of Chemical Physics,Grant/Award Numbers:DICPI202142,DICPI201938,DICPZZBS201805+1 种基金Advanced Manufacturing and Engineering Program,Grant/Award Number:A2083c0051Ministry of Education,Singapore,Grant/Award Number:MOE-MOET2EP10120-0007。
文摘The combination of super-resolution microscopy and synthetic fluorescence probes has emerged as a universal tool to monitor dynamic biological events at the nanometer scale.However,the limited site-specificity and fluorogenicity of synthetic fluorescent probes make it still difficult to realize long-term super-resolution imaging.Herein,we introduce a dynamic aggregation mediated SNAP-tag fluorogenic probe,BGAN-Aze,which can specifically bind to various SNAP-tag fusion proteins with 41-fold fluorescence enhancement.The equilibrium between the non-fluorescent aggregate/dimer(A–D)and the fluorescent monomer(M)of BGAN-Aze acts as an effective method to reduce the fluorescence background and endow BGAN-Aze with the capability of conducting washing-free super-resolution imaging of various intracellular and extracellular proteins.Using this probe,we monitored multiple dynamic biological events,such as MMC,mitophagy,the fusion of nucleolus,and the growth and contact of filopodia.We expect that BGAN-Aze will become a widely used SNAP-tag for super-resolution imaging of dynamic biological events and the A-D-M equilibrium can be a general strategy for designing fluorogenic probes.
基金supports from the National Natural Science Foundation of China (Nos. 21422606 and 21502189)Dalian Cultivation Fund for Distinguished Young Scholars (Nos. 2014J11JH130 and 2015J12JH205)
文摘Protein labeling by using a protein tag and tag-specific fluorescent probes is increasingly becoming a useful technique for the real-time imaging of proteins in living cells. SNAP-tag as one of the most prominent fusion tags has been widely used and already commercially available. Recently, various fluorogenic probes for SNAP-tag based protein labeling were reported. Owing to turn-on fluorescence response, fluorogenic probes for SNAP-tag minimize the fluorescence background caused by unreacted or nonspecifically bound probes and allow for direct imaging in living cells without wash-out steps. Thus,real-time analysis of protein localization, dynamics and interactions has been made possible by SNAP-tag fluorogenic probes. In this review,we describe the design strategies of fluorogenic probes for SNAP-tag and their applications in cellular protein labeling.
基金the National Natural Science Foundation of China(Nos.21878286,21576043,21878286)Dalian Institute of Chemical Physics,Chinese Academy of Sciences(Nos.I201938,ZZBS201805)。
文摘Monitoring dynamics of mitochondria has become an essential approach to explore the function of mitochondria in living cells with the emergence of super-resolution fluorescence microscopy.However,long-term super-resolution imaging of mitochondria is still challenging due to the lack of photostable fluorescent probes and stable mitochondria-specific markers which are not affected by the changes of mitochondrial membrane potential.Here,we introduce a method for long-term imaging mitochondrial dynamic through the SNAP-tag fluorogenic probe based on 4-azetidinyl-naphthalimide derivatives.Using structured illumination microscopy(SIM),we observed the fusion and fission of mitochondria over a course of 16 min at 109 nm resolution.Furthermore,the interactions as well as fusion between mitochondria and lysosomes were studied during mitophagy at the nanoscale.Convincingly,the combination of SNAP-tag fluorogenic probes and super-resolution fluorescence microscopy will offer a new way to monitor dynamic mitochondria in living cells.
基金supported by the National Basic Research Program of China(21077129,20877091,20890112,21125523,20921063)the National Natural Science Foundation of China(2009CB421605,2010CB933502)
文摘Methyl-CpG-binding domain(MBD)proteins can specifically recognize and bind methylated CpG sites of DNA,thus repress gene transcription.In this study,we designed and expressed two recombinant proteins,MBD2b and SNAP-MBD2b,in E.coli.An optimized protocol was developed to purify the proteins using Ni-NTA affinity cartridge and cation exchange resin.The engineered proteins purified by this method exhibited more than 93%purity and high binding avidity.We found that both SNAP-MBD2b and MBD2b were prone to aggregate during dialysis.However,this could be prevented by the use of 0.3 mol/L NaCl.The fusion of SNAP-tag with MBD2b significantly enhanced the expression of MBD2b protein in E.coli and reduced the adsorption of MBD2b on solid interfaces involved in protein purification and immobilization.The engineered proteins can be used for the study of interaction with methylated DNA and the assays for DNA methylation.
