| With the uninterrupted development and advancement of life science technology,human beings have gradually deepened their self-awareness,and people are constantly realizing that the human body is a molecular machine.The expression and interaction of various molecules in the human body are the cornerstone of molecular biology events.Therefore,it is necessary to study the structure and function of organisms at the molecular level,to clarify the essence of life phenomena and the laws of life activities,to diagnose diseases early and accurately at the molecular level,to treat diseases precisely at the molecular level.It has become a frontier research field of molecular medicine.Especially since the 21st century,cancer has become an important killer of human life and health,the development of tumor therapeutic drugs has become a vigorous research field of molecular medicine.In recent years,different therapeutic strategies have been developed for gene or protein targets based on the discovery of many biomarkers and the clear molecular mechanisms of diseases,such as gene silencing,gene editing,and protein activity inhibition or degradation.Among them,protein-targeted therapy,also known as chemotherapy,has become the main way of clinical tumor treatment.Protein-targeted drugs are mainly divided into antibody drugs and small molecule drugs.Antibody drugs are highly specific to the target and good stability,but antibodies have some disadvantages,such as difficulty in manufacturing,high cost,and lack of cell permeability.Small-molecule drugs make up for the shortcomings of antibody drugs,but the pharmacological mode driven by small-molecule drugs makes it necessary to administer high-concentration drugs for treatment,which is likely to cause toxic and side effects to normal biological tissues.In view of this,proteolytic targeting chimeras（PROTACs）have developed and changed the pharmacological mode of small-molecule drugs into an event-driven mode.PROTAC can degrade tatget proteins via regulating proteasome degradation system,thereby improving the drug efficiency.However,PROTACs require complex and precise molecular design,have no selectivity for normal cells and tumor cells,and are limited to the degradation of intracellular proteins.Therefore,the development of PROTACs has been greatly restricted.Thus,the development of simple,efficient,highly selective,and highly specific protein degraders is of great significance for tumor therapy.Nucleic acid aptamers can target targets with high specificity,high selectivity,and high affinity,and are easy to synthesize and modify.Importantly,the aptamers obtained by Cell-SELEX can selectively target cell membrane surface receptors,occupy receptor surface sites,inhibit protein activity or function,and mediate protein endocytic degradation.Therefore,aptamers are expected to be developed as tools for membrane protein degraders.This research takes full advantage of the biological functions of aptamers to develop a series of new protein degradation agents for tumor therapy.The details are as follows:（1）In Chapter 2,according to literature reports,the nucleic acid aptamer c-Met-Ap can target mesenchymal-epithelal-transition factor（c-Met）,has a competitive relationship the natural ligand hepatocyte growth factor（HGF）and inhibits the dimerization of c-Met,we further explored the biological function of c-Met-Ap aptamer.By examining the effects of c-Met-Ap on target protein levels,cell proliferation,and apoptosis,we found that c-Met-Aps can be a therapeutic agent and degrade protein.It also has a certain inhibitory effect on tumor growth.Therefore,the c-Met-Ap is expected to be developed as a high-efficiency protein degrader,which provides a new vision for the development of aptamers.（2）In Chapter 3,based on the protein degradation function of c-Met-Ap,we further explored the molecular mechanism of c-Met-Ap and determined the process of its function.The c-Met-Ap induces the phosphorylation of Y1003 site by binding to the cell membrane surface receptor,thereby recruiting c-cbl E3 ligase to interact with c-Met,causing the ubiquitination of c-Met and proteasomal degradation.Therefore,the molecular mechanism of c-Met-Ap is beneficial to the further development of protein degrader based on aptamer.（3）In Chapter 4,in order to further improve the effect of tumor therapy,we conjuated c-Met-Ap-DBCO with azide modified polyethylene glycol（m PEG-N330k Da）by click chemistry to obtain a PEGylated c-Met-Ap protein degrader,and the stability and therapeutic effect of PEGylated c-Met-Ap were explored.It was proved that the modification of PEG enhanced the resistance of c-Met-Ap to serum,and prolonged the circulation time and retention time of c-Met-Ap in vivo,enhanced the inhibitory effect on tumor growth.（4）In Chapter 5,in order to enhance the inhibitory effect on the cross-talking network signaling pathway,we ligated c-Met-Ap and 2-2t（Her2 targeting aptamer）to obtain a bivalent aptamer chimeric protein degrader（Met-Her）.The construction of bivalent aptamer chimeras enhanced the degradation of target proteins and also enhanced the inhibition of cross-talking network signaling pathways.It is expected to be developed into a universal tumor therapeutic agent. |
