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
In this work asd gene of Shigella flexneri 2a strain T32 was replaced by Vibrio cholerae toxin B subunit (ctxB) gene with DNA recombination in vivo and in vitro. The resulting derivative of T32, designed as FWL01, cou...In this work asd gene of Shigella flexneri 2a strain T32 was replaced by Vibrio cholerae toxin B subunit (ctxB) gene with DNA recombination in vivo and in vitro. The resulting derivative of T32, designed as FWL01, could stably express CtxB, but its growth in LB medium depended on the presence of diaminopimelic acid (DAP). Then form I plasmid of Shigella sonnei strain S7 was labeled with strain T32 asd gene and mobilized into FWL01. Thus a trivalent candidate oral vaccine strain, designed as FSW01, was constructed. In this candidate strain, a balanced-lethal system was constituted between the host strain and the form I plasmid expressing S. sonnei O antigen. Therefore the candidate strain can express stably not only its own O antigen but also CtxB and O antigen of S. sonnei in the absence of any antibiotic. Experiments showed that FSW01 did not invade HeLa cells or cause keratoconjunctivitis in guinea pigs. However, rabbits immunized FSW01 can elicit significant immune responses. In mice and rhesus monkey models, vaccinated animals were protected against the challenges of wild S. flexneri 2a strain 2457T and S. sonnei strain S9.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In this work asd gene of Shigella flexneri 2a strain T32 was replaced by Vibrio cholerae toxin B subunit (ctxB) gene with DNA recombination in vivo and in vitro. The resulting derivative of T32, designed as FWL01, could stably express CtxB, but its growth in LB medium depended on the presence of diaminopimelic acid (DAP). Then form I plasmid of Shigella sonnei strain S7 was labeled with strain T32 asd gene and mobilized into FWL01. Thus a trivalent candidate oral vaccine strain, designed as FSW01, was constructed. In this candidate strain, a balanced-lethal system was constituted between the host strain and the form I plasmid expressing S. sonnei O antigen. Therefore the candidate strain can express stably not only its own O antigen but also CtxB and O antigen of S. sonnei in the absence of any antibiotic. Experiments showed that FSW01 did not invade HeLa cells or cause keratoconjunctivitis in guinea pigs. However, rabbits immunized FSW01 can elicit significant immune responses. In mice and rhesus monkey models, vaccinated animals were protected against the challenges of wild S. flexneri 2a strain 2457T and S. sonnei strain S9.
