| As the boundary of the cell system,the cell membrane is responsible for responding to external biochemical signals and making corresponding cell behaviors.Proteins can be used as cell membrane surface receptors to recognize extracellular ligand molecules and activate cell signaling pathways;they can bind to membrane receptors as ligands and participate in many life processes;they can also participate in many important activities in cells,such as signaling Conduction,migration,growth and division,the localization and interaction of these proteins are critical for cellular function.Most of the current protein-based cell membrane function regulation strategies are based on genetic engineering,which is difficult to operate,time-consuming and labor-intensive in the transformation process,and difficult to guarantee the activity of the product.These problems restrict the application of proteins in the regulation of cell membrane function.With the development of functional nucleic acids and DNA nanostructures,nucleic acids have attracted more and more attention in the field of regulating cell membrane function.Nucleic acid molecules have the advantages of easy synthesis,predictability,and programmability,and are expected to become important materials for cell membrane engineering.Since the first use of enzyme-DNA complexes for nucleic acid detection in the 1980 s,various methods for synthesizing nucleic acid-protein chimeras have been developed: including non-covalent/covalent and non-site-specific/site-specific method.Using these methods to combine two biological macromolecules can integrate DNA programmability and protein versatility into a nanoscale biological device,providing new tools for the study of regulating c ell membrane function and driving cellular functions.Based on the application advantages of nucleic acid-protein chimeras,this paper uses the sequence-specific covalent linkage method to organically combine functional nucleic acid recognition function,structural DNA nanodesignability and functional proteins to construct a modular,multifunctional nucleic acid-protein chimeric platform,provides a flexible and versatile chemical approach for regulating cell membrane function and manipulating cell signal transduction.The specific research contents are as follows:(1)Construction of nucleic acid-antibody chimeras(Chapter 2).DCV protein is a HUH family endonuclease,which has the property of recognizing and cleaving target nucleic acid and covalently linking with it.This nature of covalent attachment to specific nucleic acid sequences gives flexibility and versatility in the process of synthesizing nucleic acid-protein chimeras.In this chapter,we deeply analyze the mechanism by which Bispecific T Cell Engagers(Bi TEs)mediate T cell killing of tumor cells,and recognize the importance ofαCD3 in the recognition and activation of T cells.Inspired by this,we constructed a DCV-αCD3 recombinant plasmid,prokaryotic expression and purification of DCV-αCD3 protein,and proved that DCV-αCD3 can be covalently linked to specific nucleic acid sites.We examined the effects of expression conditions,reaction buffer pH,and protein/nucleic acid ratio on covalent attachment efficiency.Based on the advantages of ea sy synthesis and diverse types of nucleic acid aptamers,we have constructed a variety of nucleic acid-antibody chimeras to lay the foundation for the development of new immunotherapy tools.(2)Modular strategy for tumor cell targeting(Chapter 3).In the previous chapter,we proposed the idea of applying nucleic acid-antibody chimeras in the field of immunotherapy.In this chapter,based on the targeted recognition properties of nucleic acid aptamers,we constructed nucleic acid aptamer×αCD3(Apt×αCD3),and further combined with the mechanism of αCD3 to activate T cells to achieve targeted killing of tumor cells by T cells.Specifically,based on the simplicity of nucleic acid sequence tailing,we added a DCV targeting recognition sequence to the 5’ end of Sgc8 aptamer to synthesize Sgc8×αCD3chimera,and verified the specificity and mediation of Sgc8×αCD3 in recognizing CEM cells.Effectiveness of T cell targeting to kill CEM cells.In addition,we also constructed a TE02×αCD3 chimera to verify its specificity in mediating T cells to kill Ramos cells,providing tools for killing different tumor cells.Meanwhile,we flexibly assembled nucleic acid aptamers to target different tumor-associated antigens,demonstrating the versatility of the Apt×αCD3 platform and developing a modular tumor targeting strategy.(3)Tunable immunotherapy with controllable avidity for tumor targeting(Chapter 4).DNA nanostructures are one-dimensional to three-dimensional fine nucleic acid nanostructures formed by hybridization of complementary DNA sequences.Branched chains of DNA nanostructures can act as scaffolds to assemble aptamers at specific locations,increasing binding affinity through polyvalent aptamers.In this chapter,two DNA nanostructures were ass embled,and a chimeric antibody-nucleic acid T-cell adaptor with adjustable valence state was constructed by taking full advantage of its addressability and the flexibility of base complementary pairing.The system includes monovalent,bivalent and multivalent CAN-TE,based on the different affinities of different numbers of nucleic acid aptamers,achieving tunable T cell immune response.In addition,in tumor xenograft model,we verified that the CAN-TE system can be used to regulate the killing effect of T cells,including inhibiting tumor growth,regulating cytokine secretion,and the degree of T cell infiltration in tumor tissue.Based on the facile fabrication of DNA nanostructures to display different number of the nucleic acid aptamers,the multivalent CAN-TE structure combines different types of nucleic acid aptamers on both sides,which can solve the problem of antigen escape in the process of immunotherapy and further expand the the application of nucleic acid-antibody chimeras in the field of immunotherapy.This chapter provides a valence-tunable nucleic acidantibody chimera system for regulating the binding affinity between T cells and tumor cells,providing a novel tool for on-demand modulation of immunotherapy effects.(4)Artificial control over the intracellular signal transduction based on nucleic acid-protein chimera regulation of protein translocation(Chapter 5).As the key executive molecules of cellular life activities,the spatiotemporal localization of proteins is crucial for regulating cellular functions.In this chapter,a nucleic acid-protein chimera system for regulating intracellular protein localization was constructed based on the property of covalently linking DCV proteins to targeted nucleic acid sequences,using inverted spheri cal nucleic acids as tools.After the reverse spherical nucleic acid is fused with the cell,the nucleic acid is localized inside the cell membrane,and then the nucleic acid specifically induces the translocation of its complementary nucleic acid and generates a membrane localization signal.In addition,we transfected the EGFP-DCV recombinant plasmid into cells,and through laser confocal experiments,proved that the nucleic acid inside the membrane can be used to regulate intracellular protein localization.Since protein localization affects cell signaling,we constructed m Cherry-DCV-iSH recombinant plasmids to confirm that nucleic acid-protein covalent linkage can be used to regulate the translocation of iSH protein and Akt-PH protein to the cell membrane,thereby activating the intracellular PI3 K signaling pathway.It further expands the application scope of nucleic acid-protein chimeras and provides new tools for artificial regulation of cell functions. |
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