Lipid rafts(LRs)are relatively well-ordered functional microdomains in cell membranes and play an irreplaceable role in physiological processes as a transduction platform for multiple signaling pathways.Due to their s...Lipid rafts(LRs)are relatively well-ordered functional microdomains in cell membranes and play an irreplaceable role in physiological processes as a transduction platform for multiple signaling pathways.Due to their small size and high spatiotemporal dynamics,it is difficult to perform lipid raftlocalized biomolecule imaging on the surface of living cells.Here,we report a DNA nanotechnology-based platform for reversible manipulation and localized analysis of lipid rafts,which consists of two modules:“patching and coding probe pair”and“fishing probe”.The probe pair is generated by modifying two different sets of connectable DNA structures on a lipid raft-specific protein.After recognizing lipid rafts,the two probes in close proximity are linked by a DNA ligase reaction to form a lipid raft identity(LRID)code.The LR-ID strand patches and stabilizes the lipid raft structure.Interestingly,the raft patches formed can be depatched by restriction endonucleases,providing the first reversible manipulation of the lipid raft structure in living cells.We also designed a“fishing probe”with a DNA hairpin structure using an aptamer that can specifically bind to the target.The probe can cascade the reaction to two input signals“LR-ID”and“target protein”to generate an“off-on”fluorescence switch,allowing imaging and dynamic monitoring of target proteins localized in lipid rafts.By encoding arbitrary targets(in the case of glycans)in lipid rafts,we have created a universal lipid raft-localized imaging platform.This work provides an integrated analytical and manipulative platform to reveal lipid rafts and associated signaling pathways at the molecular level.展开更多
Historically constrained by the low throughput of traditional Time-Correlated Single-Photon Counting(TCSPC),a new method utilizes an optimized,time-gated single-photon camera acquisition scheme for achieving single-mo...Historically constrained by the low throughput of traditional Time-Correlated Single-Photon Counting(TCSPC),a new method utilizes an optimized,time-gated single-photon camera acquisition scheme for achieving single-molecule lifetime precision comparable to TCSPC,but across a wide field of view simultaneously.This high-throughput capability transforms single-molecule FLIM from a specialized,time-consuming effort into a scalable,quantitative assay.The resulting multimodal imaging platform provides unprecedented simultaneous insight into the nanoscale location(SMLM)and local environment(FLIM)of biological molecules,advancing our ability to map complex biophysical parameters like FRET efficiency in living systems.展开更多
基金support from the National Natural Science Foundation of China(21974067,22274073)the Fundamental Research Funds for the Central Universities(020514380309,021414380502,2022300324)the State Key Laboratory of Analytical Chemistry for Life Science(5431ZZXM2305,5431ZZXM2204).
文摘Lipid rafts(LRs)are relatively well-ordered functional microdomains in cell membranes and play an irreplaceable role in physiological processes as a transduction platform for multiple signaling pathways.Due to their small size and high spatiotemporal dynamics,it is difficult to perform lipid raftlocalized biomolecule imaging on the surface of living cells.Here,we report a DNA nanotechnology-based platform for reversible manipulation and localized analysis of lipid rafts,which consists of two modules:“patching and coding probe pair”and“fishing probe”.The probe pair is generated by modifying two different sets of connectable DNA structures on a lipid raft-specific protein.After recognizing lipid rafts,the two probes in close proximity are linked by a DNA ligase reaction to form a lipid raft identity(LRID)code.The LR-ID strand patches and stabilizes the lipid raft structure.Interestingly,the raft patches formed can be depatched by restriction endonucleases,providing the first reversible manipulation of the lipid raft structure in living cells.We also designed a“fishing probe”with a DNA hairpin structure using an aptamer that can specifically bind to the target.The probe can cascade the reaction to two input signals“LR-ID”and“target protein”to generate an“off-on”fluorescence switch,allowing imaging and dynamic monitoring of target proteins localized in lipid rafts.By encoding arbitrary targets(in the case of glycans)in lipid rafts,we have created a universal lipid raft-localized imaging platform.This work provides an integrated analytical and manipulative platform to reveal lipid rafts and associated signaling pathways at the molecular level.
文摘Historically constrained by the low throughput of traditional Time-Correlated Single-Photon Counting(TCSPC),a new method utilizes an optimized,time-gated single-photon camera acquisition scheme for achieving single-molecule lifetime precision comparable to TCSPC,but across a wide field of view simultaneously.This high-throughput capability transforms single-molecule FLIM from a specialized,time-consuming effort into a scalable,quantitative assay.The resulting multimodal imaging platform provides unprecedented simultaneous insight into the nanoscale location(SMLM)and local environment(FLIM)of biological molecules,advancing our ability to map complex biophysical parameters like FRET efficiency in living systems.
