Rolling Cycle Replication Technology Based Cell Assembly And Its Application In Regulating Cell Function

Cell assembly usually is conducted in a pre-designed way to realize specific cell-to-cell connections,thereby giving the assembled cell population special functions.Constructing cell assembly models is not only critical for studying cell-cell communication,tissue and organ formation,immune response and cancer metastasis,but also for reconstructing in vitro disease models and studying the effectiveness of various treatment methods.However,the traditional monovalent modification method has low cell assembly efficiency,which greatly restricts the wide application of cell assembly technology.The traditional monovalent modification provides only one binding site at each modification site,while the advantage of polyvalent modification is that it can expand multiple binding sites at each modification site.This thesis is based on the scientific problem of how to achieve efficient cell assembly.Using the precise base pairing principle and programmability of DNA molecules,the long-chain DNA with repetitive sequence structure synthesized by rolling circle replication technology realizes the polyvalence modification of cell surface and improves cell assembly efficiency.This paper introduces two multivalent modification methods to improve cell assembly efficiency.It consists of the following two parts:Part 1:Rolling circle replication technology for cell assembly and its application in re-education of macrophages.Rolling circle replication technology was used to prepare long single-stranded DNA with repetitive sequences for modifying the cell membrane.Free sulfhydryl groups were obtained through mild reduction of the cell membrane.The Michael addition reaction between the sulfhydryl group and maleimide is used to modify the maleimide-terminated long-chain DNA on the cell membrane.Then the efficient assembly between specific cells was achieved by complementary base pairing,with an obvious increase of cell assembly efficiency to 42.62%.At the same time,we applied this cell assembly strategy to investigate the re-education effect of M1 type macrophages on M2 type macrophages.Part 2:"Click"reaction combining with rolling circle replication technology for efficient cell assembly.reaction is efficient to achieve cell assembly.One target cell was incorporated with the N₃ group modified by glycol-metabolic strategy.Meanwhile,another cell is treated with the long chain DNA modified with dibenzocyclootyne?DBCO?by rolling cycle replication.The N₃ group and DBCO group can quickly form a covalent bond through the"Click"reaction to achieve efficient cell assembly,and the efficiency of cell assembly is as high as 66.97%.At the same time,the cell assembly technology can be used to inhibit cell metastasis.