Bioorthogonal cleavage and ligation reactions together form one more integrated system about the repertoire of bioorthogonal chemistry,capacitating an array of thrilling new biological applications.The bond-cleavage t...Bioorthogonal cleavage and ligation reactions together form one more integrated system about the repertoire of bioorthogonal chemistry,capacitating an array of thrilling new biological applications.The bond-cleavage type and position of biomolecular remain a great challenge,which determines the metabolic pathway of the targets in living systems.Herein we designed two linkages of methylene and carbonyl group attached the N-3 position of the 5-ethynyl-2’-deoxyuridine(EdU)base or the oxygen atom at deoxyribose 3’position to a photocaging group,which would be cleaved by irradiation with 365 nm ultraviolet light.EdU derivatives linked by methylene at the N-3 position had better photodecage efficiency and stability in the absence of light.This paper provides a strategy for studying the nucleoside metabolic pathways in cells,which can easily and conveniently evaluate the effect of the position and type of the linkages.The developed strategy affords a reference for controlling spatial and temporal metabolism of small-molecule drugs,allowing direct manipulation of intact cells under physiological conditions.展开更多
RNA labeling is vital for the study of an RNA structure,cellular distribution,localization,and metabolism.Herein,we report N6 cyclopropane-modified adenosine(cpA)as a new analog for metabolic RNA labeling.We successfu...RNA labeling is vital for the study of an RNA structure,cellular distribution,localization,and metabolism.Herein,we report N6 cyclopropane-modified adenosine(cpA)as a new analog for metabolic RNA labeling.We successfully applied inverse electrondemand Diels–Alder(iEDDA)chemistry to label cellular RNA with cpA.This labeling technique is practical and provides a new platform to study RNA roles in cells in a metal-free manner.This simple and robust assay represents a significant advancement in the profiling methods of the nascent transcriptome using chemical approaches.展开更多
Main observation and conclusion Bioorthogonal click chemistry has emerged as a powerful tool for the specific modification of proteins in complex mixtures.Metabolic labeling of proteins with azide followed by the copp...Main observation and conclusion Bioorthogonal click chemistry has emerged as a powerful tool for the specific modification of proteins in complex mixtures.Metabolic labeling of proteins with azide followed by the copper-catalyzed azide−alkyne cycloaddition(CuAAC)with alkyne-based affinity probes/beads is widely applied to study protein turnover and post-translational modifications(PTMs).展开更多
The integration of RNA metabolic labelling by nucleoside analogues with high-throughput RNA sequencing has been harnessed to study RNA dynamics.The immunoprecipitation purification or chemical pulldown technique is ge...The integration of RNA metabolic labelling by nucleoside analogues with high-throughput RNA sequencing has been harnessed to study RNA dynamics.The immunoprecipitation purification or chemical pulldown technique is generally required to enrich the analogue-labelled RNAs.Here we developed an a^(6)A-seq method,which takes advantage of N^(6)-allyladenosine(a^(6)A)metabolic labelling on cellular mRNAs and profiles them in an immunoprecipitation-free and mutation-based manner.a^(6)A plays a role as a chemical sequencing tag in that the iodination of a^(6)A in mRNAs results in 1,N^(6)-cyclized adenosine(cyc-A),which induces base misincorporation during RNA reverse transcription,thus making a^(6)A-labelled mRNAs detectable by sequencing.A nucleic acid melting assay was utilized to investigate why cyc-A prefers to be paired with guanine.a^(6)A-seq was utilized to study cellular gene expression changes under a methionine-free stress condition.Compared with regular RNA-seq,a^(6)A-seq could more sensitively detect the change of mRNA production over a time scale.The experiment of a^(6)Acontaining mRNA immunoprecipitation followed by qPCR successfully validated the high-throughput a^(6)A-seq data.Together,our results show a^(6)A-seq is an effective tool to study RNA dynamics.展开更多
A novel metabolic chemical reporter of Ac_(3)6deo Glc NAz was developed and confirmed as an effective probe for O-Glc NAc modification. Ac_(3)6deo Glc NAz labeling predominantly occurs in intracellular OGlc NAcylated ...A novel metabolic chemical reporter of Ac_(3)6deo Glc NAz was developed and confirmed as an effective probe for O-Glc NAc modification. Ac_(3)6deo Glc NAz labeling predominantly occurs in intracellular OGlc NAcylated proteins rather than cell-surface glycoproteins. Of note, it could reduce the artificial S-glycomodification compared to Ac_(4)Gal NAz and Ac_(4)Glc NAz. This new reporter allows to be widely used in the field of proteomic identification of O-Glc NAcylation.展开更多
Exosomal glycoproteins play significant roles in many physiological and pathological procedures. However, the current methods for studying exosomal glycoproteins have low sensitivity or can affect exosomal biological ...Exosomal glycoproteins play significant roles in many physiological and pathological procedures. However, the current methods for studying exosomal glycoproteins have low sensitivity or can affect exosomal biological function. Herein, we developed a proximity dual-tagging strategy using an induced hybridization chain reaction(HCR) from the target’s non-functional epitope for amplified visualization and functional exploration of exosomal protein-specific glycosylation. This strategy leverages dualtagging based on the aptamer with little influence on target function and metabolic glycan labelling, and the rigid product and high sensitivity of HCR. The method improves the signal of visualizing exosomal PD-L1(exo PD-L1) by 7.7-fold compared with the signal without HCR amplification without affecting the natural exo PD-L1/PD-1 interaction. As a result, we verified that the interaction between exo PD-L1 and PD-1 positive cells is positively correlated to the glycosylation level of exo PD-L1. Overall,we have developed a sensitive method with little functional influence to visualize exosomal protein-specific glycosylation in situ,offering a powerful tool for studying the biological implications of exosomal glycoproteins.展开更多
基金supported by the National Natural Science Foundation of China(Nos.21432008,91753201 and 21721005)the large-scale instrument and equipment sharing foundation of Wuhan University。
文摘Bioorthogonal cleavage and ligation reactions together form one more integrated system about the repertoire of bioorthogonal chemistry,capacitating an array of thrilling new biological applications.The bond-cleavage type and position of biomolecular remain a great challenge,which determines the metabolic pathway of the targets in living systems.Herein we designed two linkages of methylene and carbonyl group attached the N-3 position of the 5-ethynyl-2’-deoxyuridine(EdU)base or the oxygen atom at deoxyribose 3’position to a photocaging group,which would be cleaved by irradiation with 365 nm ultraviolet light.EdU derivatives linked by methylene at the N-3 position had better photodecage efficiency and stability in the absence of light.This paper provides a strategy for studying the nucleoside metabolic pathways in cells,which can easily and conveniently evaluate the effect of the position and type of the linkages.The developed strategy affords a reference for controlling spatial and temporal metabolism of small-molecule drugs,allowing direct manipulation of intact cells under physiological conditions.
基金supported financially by the National Natural Science Foundation of China(21432008,91753201,and 21721005).
文摘RNA labeling is vital for the study of an RNA structure,cellular distribution,localization,and metabolism.Herein,we report N6 cyclopropane-modified adenosine(cpA)as a new analog for metabolic RNA labeling.We successfully applied inverse electrondemand Diels–Alder(iEDDA)chemistry to label cellular RNA with cpA.This labeling technique is practical and provides a new platform to study RNA roles in cells in a metal-free manner.This simple and robust assay represents a significant advancement in the profiling methods of the nascent transcriptome using chemical approaches.
基金This work was supported by grants from China State Key Basic Research Program Grants(2016YFA0501403,2016YFA0501404 and 2020YFE0202200)the National Natural Science Foundation of China(31700088)+1 种基金Guangdong Provincial Fund for Distinguished Young Scholars(2019B151502050)Shenzhen Innovation of Science and Technology Commission Grant(JCYJ20170412154126026).
文摘Main observation and conclusion Bioorthogonal click chemistry has emerged as a powerful tool for the specific modification of proteins in complex mixtures.Metabolic labeling of proteins with azide followed by the copper-catalyzed azide−alkyne cycloaddition(CuAAC)with alkyne-based affinity probes/beads is widely applied to study protein turnover and post-translational modifications(PTMs).
基金the National Key R&D Program of China(2022YFA1103702 and 2017YFA0506800)the National Natural Science Foundation of China(22022702 and 21977087)the Fundamental Research Funds for the Central Universities,and MOE Key Laboratory of Macromolecular Synthesis and Functionalization,Zhejiang University(2022MSF04).
文摘The integration of RNA metabolic labelling by nucleoside analogues with high-throughput RNA sequencing has been harnessed to study RNA dynamics.The immunoprecipitation purification or chemical pulldown technique is generally required to enrich the analogue-labelled RNAs.Here we developed an a^(6)A-seq method,which takes advantage of N^(6)-allyladenosine(a^(6)A)metabolic labelling on cellular mRNAs and profiles them in an immunoprecipitation-free and mutation-based manner.a^(6)A plays a role as a chemical sequencing tag in that the iodination of a^(6)A in mRNAs results in 1,N^(6)-cyclized adenosine(cyc-A),which induces base misincorporation during RNA reverse transcription,thus making a^(6)A-labelled mRNAs detectable by sequencing.A nucleic acid melting assay was utilized to investigate why cyc-A prefers to be paired with guanine.a^(6)A-seq was utilized to study cellular gene expression changes under a methionine-free stress condition.Compared with regular RNA-seq,a^(6)A-seq could more sensitively detect the change of mRNA production over a time scale.The experiment of a^(6)Acontaining mRNA immunoprecipitation followed by qPCR successfully validated the high-throughput a^(6)A-seq data.Together,our results show a^(6)A-seq is an effective tool to study RNA dynamics.
基金supported by the National Natural Science Foundation of China (Nos. 81901622, 31770987, 81971497 and 21907022)Key R&D and Promotion Special Project in Henan Province (Nos. 212102310185, 212102310899)+1 种基金Key Scientific Research Projects in Henan Colleges and Universities (No. 20A350001)Joint Construction Project of Henan Medical Science and Technology Project (No. LHGJ20210566)。
文摘A novel metabolic chemical reporter of Ac_(3)6deo Glc NAz was developed and confirmed as an effective probe for O-Glc NAc modification. Ac_(3)6deo Glc NAz labeling predominantly occurs in intracellular OGlc NAcylated proteins rather than cell-surface glycoproteins. Of note, it could reduce the artificial S-glycomodification compared to Ac_(4)Gal NAz and Ac_(4)Glc NAz. This new reporter allows to be widely used in the field of proteomic identification of O-Glc NAcylation.
基金supported by the National Natural Science Foundation of China (22022409, 21735004, 21874089)the Program for Changjiang Scholars and Innovative Research Team in University(IRT13036)+1 种基金the National Science Fund for Fostering Talents in Basic Science (J1310024)XMU Training Program of Innovation and Entrepreneurship for Undergraduates。
文摘Exosomal glycoproteins play significant roles in many physiological and pathological procedures. However, the current methods for studying exosomal glycoproteins have low sensitivity or can affect exosomal biological function. Herein, we developed a proximity dual-tagging strategy using an induced hybridization chain reaction(HCR) from the target’s non-functional epitope for amplified visualization and functional exploration of exosomal protein-specific glycosylation. This strategy leverages dualtagging based on the aptamer with little influence on target function and metabolic glycan labelling, and the rigid product and high sensitivity of HCR. The method improves the signal of visualizing exosomal PD-L1(exo PD-L1) by 7.7-fold compared with the signal without HCR amplification without affecting the natural exo PD-L1/PD-1 interaction. As a result, we verified that the interaction between exo PD-L1 and PD-1 positive cells is positively correlated to the glycosylation level of exo PD-L1. Overall,we have developed a sensitive method with little functional influence to visualize exosomal protein-specific glycosylation in situ,offering a powerful tool for studying the biological implications of exosomal glycoproteins.
