New Directed Evolution Strategies And Biomedical Applications Of Aptamers

Molecular recognition is the most basic and ubiquitous chemical event in biological systems.In recent years,as an important molecular recognition probe,aptamers have proven to be indispensable tools in the field of biomedicine,which can be used in immunotherapy response prediction,tumor detection and analysis,virus neutralization and inhibition.However,with the deepening of related research,researchers have encountered some new research challenges.For example,how to obtain aptamers with high-sensitivity,high-performance,controllable structure and function,and how to obtain aptamers efficiently still are the central tasks and important challenges in the field of aptamers.In response to this task and challenge,this thesis focuses on the development of new and efficient aptamer directed evolution strategies and their application in biomedicine,and intends to carry out the following work:1.Homogeneous,Low-volume,Efficient and Sensitive Quantitation of Circulating Exosomal PD-L1 for Cancer Diagnosis and Immunotherapy Response PredictionImmunotherapy has brought revolutionary changes to cancer treatment,but its efficacy has been seriously hindered due to the lack of effective predictors.For this reason,a homogeneous,low-volume,efficient and sensitive exosomal PD-L1 quantitation method(HOLMES-ExoPD-L1)was established,which can be used in cancer diagnosis and immunotherapy response prediction.PD-L1 protein was highly glycosylated,HOLMES-ExoPD-L1 uses aptamer obtained by directional evolution of non-post-translational modified PD-L1 proteins as recognition molecules,the aptamer recognizes the bared polypeptide antigens rather than the post-translational modifications proteins,so it was easy to pass through glycosylated PD-L1,to improve PD-L1 recognition efficiency;At the same time,homogeneous thermophoresis with fast binding kinetics was used for detection to improve detection sensitivity.The free aptamers and the exosome-aptamer complex have different distributions in the temperature field induced by thermophoresis so that exosomal PD-L1 can be quantitatively detected without separation and washing.Therefore,the expression level of circulating exosomal PD-L1 detected by HOLMES-ExoPD-L1 can effectively distinguish cancer patients from healthy volunteers,and for the first time was found to correlate positively with the metastasis of adenocarcinoma.Compared with the enzyme-linked immunosorbent assay(ELISA)-based methods for detecting exosomal PD-L1,HOLMES-ExoPD-L1 improves the detection sensitivity,saves time and easy to operate.Therefore,HOLMES-ExoPD-L1 provides a new method and a new idea for early cancer diagnosis and immunotherapy response prediction.2.In Situ Visualization of PD-Ll-Specific Glycosylation on Tissue BiopsiesTo realize the visualization of PD-L1-specific glycosylation,based on the glycosylation-free aptamer of PD-L1 obtained by directed evolution,we developed an in situ visualization method,FLAG(a lectin for glycan labelling and the aptamer for PD-L1 polypeptide antigen,offering visualization of PD-L1-specific glycosylation).This method can be used to observe protein-specific glycosylation on tissue slides in situ by exploiting the advantages of PD-L1 aptamer with small steric hindrance,together with the metabolism-free lectin labeling.The FLAG strategy has been used to visualize the states and distribution of PD-Ll-specific glycosylation on lung cancer tissue slides.This straightforward and non-metabolically labeled strategy was not only limited to scientific research but also could be easily generalized to clinical practice.Therefore,the FLAG strategy provides a powerful tool to reveal the significance of PDL1 glycosylation in diagnosis,which is expected to shed new light on the development of immune checkpoint inhibitors and identify new biomarkers to predict patients who would benefit from immune checkpoint therapy.Moreover,the FLAG strategy can be applied in a wide variety of areas by simply replace the type of aptamer or ligand.3.Molecular Crowding Evolution for Enabling Discovery of Enthalpy-Driven Aptamers for Robust Biomedical ApplicationsThe enthalpy-driven aptamer is an ideal probe in a complex environment,which has high affinity and selectivity in biomedical applications.However,there is still a lack of methods for directly discovering enthalpy-driven aptamers.Therefore,a new molecular crowding screening method,molecular crowding SELEX was developed to direct evolutionary enthalpy-driven aptamers.Aiming at the tumor biomarker EpCAM protein,three aptamers were successfully evolved,and all of them were proved to be enthalpy-driven by thermodynamic analysis,which established the feasibility of finding enthalpy-driven aptamers effectively by molecular crowding SELEX.Further comparing the aptamer evolved in traditional SELEX(SYL-3C aptamer)and molecular crowded SELEX(SYL-H2C aptamer),it was found that the affinity of SYL-H2C was 6.5 times higher than that of SYL-3C.With its improved thermodynamic properties,enthalpy-driven S YL-H2C aptamers can detect circulating tumor cells in blood samples of cancer patients with excellent detection accuracy(10/10).Therefore,the proposed molecular crowding SELEX provides a promising direction for the discovery of robust binding probes in biomedicine.4.Activation of Aptamers with Gain-of-Function by Small-Molecule-Clipping of Intramolecular MotifsTo obtain aptamers with controllable structure and function,we developed the concept of "clipped aptamer" and its directed evolution process "Clipped Aptamer SELEX".Through the direct evolution of "Clipped Aptamer SELEX",the "clipped aptamer" targeting the tumor biomarker EpCAM protein was obtained."Clipped aptamer" makes use of the specific recognition between the DNA mismatch binding molecular glue(Z-NCTS)and the preset CGG/CGG site in the DNA sequence,which can quickly "clip" the two CGG sites in the DNA sequence and initiate the efficient transition from binding inactive state to an active state under ideal thermodynamic conditions.Through experiments and molecular docking simulation,the results show that "clipped aptamer" can indeed regulate its three-dimensional structural changes through Z-NCTS,and achieve an efficient transition from the binding inactive state to an active state,thus accurately regulating cellular adhesion.Therefore,"clipped aptamer" has great potential in biosensor、imaging、cell behavior regulation and drug delivery.In theory,this screening method can directly evolve conformational regulatory aptamers of any target,avoids the trial and error of sequence design in conventional methods,and provides a new idea for the study of constructing aptamer with controllable structure and function.5.Bispecific neutralizing aptamer inhibits SARS-CoV-2 virus infectionThe variation of SARS-CoV-2 leads to the enhancement of infectious ability,there is an urgent need to develop neutralizing agents against SARS-CoV-2 and the associated mutant.To further improve the ability of aptamer to inhibit the infection of SARS-CoV-2 and the associated mutant,the bispecific neutralizing aptamer was designed by DNA self-assembly to synergistically block the interaction of SARS-CoV2 receptor-binding domain(RBD)and angiotensin-converting enzyme-2(ACE2),leading to inhibit SARS-CoV-2 infection.Taking advantage of the simultaneous recognition of di-heterogenous aptamers,the bispecific neutralizing aptamer has an enhanced affinity to the SARS-CoV-2RBD(a dissociation constant value of 4.8 nM)and a potent neutralization effect for pseudotyped SARS-CoV-2(a half-maximal inhibitory concentration of 8.2 nM).Due to the good affinity,biosafety,stability and non-immunogenic,the bispecific neutralizing aptamer has a flexible neutralizing effect and vastly capable of neutralizing pseudotyped SARS-CoV-2 mutants.Besides,the bispecific neutralizing aptamer can be extended to nanodevices such as DNA tetrahedron、icosahedron and gold nanoparticle as monomer building blocks,to further improve the ability to inhibit SARS-CoV-2 infection.Therefore,the bispecific neutralizing aptamer blocking strategy provides a new direction for developing therapeutic agents against COVID-19.