Aptamer-based Recognition And Regulation Of Membrane Proteins On Living Cells

Cell membrane receptor are important mediators between cells and their external environment,and play an important role in cont rol of many biological processes such as cell cycle,proliferation,communication,migration and apoptosis.For example,T cell activation is initiated by the specific antigen recognition of T cell surface receptor(TCR),and the magnitude and quality of i ts activity ultimately depend on the balance regulation of co-stimulatory and co-inhibitory receptor.Therefore,the study on the regulation of the functional activity of immune-related membrane receptors was of great significance to the understanding of t he law of life,the elucidation of signal transduction mechanism,and the research of disease diagnosis and treatment.In the past few decades,methods based on small molecules have become one of the main strategies to study membrane receptor-related signaling pathways.However,small molecule compounds tended to cause off-target problems due to their low selectivity.To further increase the specificity of receptor regulation,researchers have applied optogenetics to the study of membrane receptors.Although optogenetics achieved spatiotemporal resolution of membrane receptor activity,it changed the structure and expression form of the target protein,which might bring unpredictable interference to the study of related biological processes.Therefore,it was urgent to develop simple,convenient,universal and specific methods to regulate membrane receptor activity.Since the advent of aptamer known as "chemical antibodies" in 1990,they have attracted wide attention in the fields of chemical sensing,molecul ar imaging,disease diagnosis and treatment,target delivery and other fields due to their excellent properties(high specificity,high affinity,easy synthesis and modification,low immunogenicity,etc.).The combination of DNA nanotechnology with atomic precision localization and structural reconfiguration would provide infinite possibilities for aptamer to accurately manipulate membrane receptor activity at the nanoscale.Based on the above research background,focusing on how to develop simple,convenient,universal and specific methods to regulate membrane receptor activity,this work constructed membrane receptor recognition and activity regulation platforms based on aptamer,and explore d its application in T cell immune regulation and anti-tumor application.The research contents were as follows:(1)The development of membrane receptor recognition probes with excellent recognition performance was a prerequisite for the regulation of their activity.In Chapter 2,aiming at the scientific problem that a ptamers were easily degraded by nucleases,we developed environment-responsed aptamer stabilization strategy by introducing a partial fragment of ATP aptamer into the end of aptamer Sgc8c(called ARP-sgc8c),in which the binding of ATP molecules could prot ect the DNA sequence from exonuclease degradation,thereby improving the biostability of ARP-sgc8 c in the tumor microenvironment.We demonstrated that the introduction of ATP aptmers did not affect the molecular recognition ability of Sgc8 c.At the same time,the improvement of the stability of ARP-sgc8 c was proved by enzyme digestion and serum stability experiments.We also applied the design to improve the stability of aptamers such as XQ-2d.Further,the feasibility of this strategy was investigated by in vivo mouse imaging experiments.(2)In order to achieve customized regulation of cell membrane receptor signal strength,given that the molecular distance between dimer receptors might be another important factor affecting the output strength of downstre am signal cascades.In Chapter 3,we selected CD28,a co-stimulatory receptor of T cells,as the protein model,and successfully constructed aptamer-functionalized,tweezer-like DNA nanodevices(Apt-NT),aiming to regulate the strength of downstream signal by changing the lateral distance of CD28 receptor.By investigating the formation of Apt-NT in different states and its ability to regulate T cell activity,we found that the lateral distance between CD28 receptors was inversely proportional to the activa tion degree of T cells.Further,we demonstrated that Apt-NT could realize reversible transformation between closed and open states,thus realizing the custom regulation of T cell activity.In addition,we extended this design to the regulation of c-Met receptor activity,and verified the ability of Apt-NT to dynamically regulate c-Met receptor through protein expression and cell behavior experiments.(3)In order to further expand the regulation of protein activity based on membrane receptor specific recognition,in Chapter 4,we selected cancer cells and T cells as model cells,designed and constructed aptamer-functionalized DNA circuit to achieve target cell-responsed cell membrane receptors regulation.The DNA circuit was composed of recognition-then-triggering and aggregation-then-activation modules.The trigger probe AS-Tr could sense the existence of cancer cell-specific membrane receptors,then released Tr and initiated hybridization chain reaction to regulate the state of CD28 protein on T cell memb rane.This process fully simulated the behavior of signal perception,processing and transmission in the intercellular communication network.We optimized AS-Tr probes by flow cytometry and confocal imaging.Furthermore,the non-specific leakage caused by AS-Tr was investigated and the leakage reasons were speculated.At the same time,we demonstrated the specific binding of the aggregation-then-activation module to T cells and the ability to regulate the state of the CD28 receptor.(4)Based on the research basis of Chapter 4,in order to explore the application prospect of membrane receptor activity regulation on cell-cell interaction and anti-tumor immunity.In Chapter 5,we addressed the problem that CAR T cells were to some extent affected by complex a nd potentially risky genetic modification processes.We applied the aptamer-functionalized DNA circuit to the regulation of T cell and cancer cell interactions,aiming to establish a new mode of interaction between naive T cells and cancer cells without in volving gene editing.We demonstrated that only when the target cancer cells were present,the logic operation of the DNA circuit would up-regulate the activity of T cells.Furthermore,we constructed model tumor cell line expressing the target protein by lentivirus transfection,through in vitro and in vivo experiments proved that when only target cancer cells exist,the operation of the DNA logic circuit would enhance the activity of T cells and improve the ability of T cells to kill tumor cells.