Application Of DNA Chemical Reaction Network In Cell Function Regulation And Cell Imaging

As a kind of genetic material,DNA is widely used in genetics related research.With the discovery of nucleic acids with independent structure and specific non-genetic biological functions and the development of DNA nanotechnology,DNA has been widely used as a functional material in the fields of biosensor,drug delivery and so on.With the in-depth study of DNA strand displacement reaction,dynamic DNA nanostructures have become a very powerful tool.Researchers can design complex network behaviors based on DNA dynamic nanostructures to manipulate the temporal and spatial distribution of matter at the molecular level.The regulation of cell behavior is mostly realized by controlling the receptors or ligands on the surface of cell membrane.However,there are some limitations in the method of ligand regulation based on genetic engineering,such as high technical complexity,potential toxicity and so on.DNA dynamic nanotechnology provides a powerful tool for dynamic control of surface receptors on the nanoscale,and has an important application prospect in the artificial regulation of cell behavior.Therefore,DNA has become a new practical tool in the field of cell regulation.However,most of the current methods of using DNA to regulate cell behavior are realized through simple one-way and one-step reaction,while biological reaction is more often a complex chemical reaction network.In order to better understand these biological reactions and achieve human regulation of more complex systems,we need to design appropriate methods to achieve more complex human defined regulation at the cellular level.In order to regulate the cellular level reaction network,we combine the DNA chemical reaction network with the cell based on the functional nucleic acid and DNA strand displacement reaction,and finally achieve the regulation of cell behavior.Considering the difficulty of realizing complex DNA chemical reaction network,we select three kinds of chemical reaction network(cascade?logic?and amplification)as representatives,and construct scalable DNA chemical reaction network based on functional nucleic acid and DNA strand displacement reaction.And It is applied to the fields of cell behavior regulation and secretory factor detection.The specific research content is as follows:1.A DNA cascade reaction network that can regulate the signal exchange between cells was constructed.We first constructed an extensible DNA strand displacement module which was composed of binding part,functional part and toehold part.We built a four-stage DNA cascade reaction by rationally combining the modules.According to the kinetic results of the cascade reaction of different stages,we have proved that the artificially input signal is indeed transmitted backward step by step through the cascade reaction.Subsequently,we used functional nucleic acids to connect different modules of the DNA cascade network on different groups of Hep G2 cells.The results of the study showed that although there was a certain loss in the transmission of this signal on the cells,it was possible to realize the gradual transmission of artificial control signals between two groups of cells;2.A DNA logic response network that can realize programmable cell screening in a multi-cell system was constructed.And It was applied to regulate the activation of cell signaling pathways.We first constructed and optimized two different logic networks in vitro,AND and NIMPLY.After inputting two different signals,the logic network can respond to external signals according to artificially designed logic and unblock the output DNA strand to achieve fluorescence quenching.Taking the AND logic network as a representative,we used the specific binding function of DNA aptamer to logically bind the AND logic network to cells which expressing two cell membrane surface receptors,MUC1 and Ep CAM.Moreover,the DNA strand displacement reaction only occured on MCF-7 cells which expressing both receptors to release our pre-blocked HGF protein.It proveed that we could achieve only the logical behavioral regulation of MCF-7 cells in a multi-cell system;3.The HCR-based in situ detection and imaging amplification response network of TNF? was constructed.We first constructed and verified the amplification effect of the HCR amplification reaction network in vitro.The experiment proved that it could be amplified nearly 5 times compared with the control.Taking the detection of TNF?as a representative,we designed a DNA hairpin probe and its corresponding HCR amplification network.After optimizing the sequence of the HCR amplification network,we proved that the HCR amplification reaction network could achieve a signal-to-background ratio of more than 2 times compared with the DNA hairpin probe during detection.Subsequently,we used Jurkat cells and HT1080 cells to culture the cell tumor spheres,and used the CBD-DCV recombinant protein which was constructed by our group to combine the DNA hairpin probe and HCR amplification network with the tumor spheres.The secretion of TNF? is detected in real-time in situ.The results proved that the HCR amplification network could achieve more than 2times compared with DNA probes in the tumor spheres system.