Research On The Construction Of Covalent Aptamers And Their Applications In Disease Diagnosis And Treatment

As novel molecular recognition elements,aptamers are promising tools for targeted imaging or treatment of various diseases.Aptamer-based disease detection and therapeutic approaches have made substantial progress in cost control and clinical practice.However,the conformational instability of aptamers usually leads to the dissociation of aptamer-target complexes under harsh washing and other environmental stresses,resulting in reduced detection sensitivity and diminished therapeutic efficacy.Covalent aptamers are a class of aptamers that can interact with specific targets and form covalent bonds.Covalent aptamers can selectively cross-link with their targets by using proximity effects to bring them closer to the targets.Due to the formation of covalent bonds,the binding between aptamers and targets is irreversible,which can increase the stability and affinity of aptamers for target recognition.Covalent bonds are not easily broken,making aptamer-target covalent complexes more stable than conventional non-covalent complexes.Covalent aptamers can be used to selectively bind targets from complicated biological samples,thereby enabling target purification and enrichment and improving target selectivity.Covalent aptamers are expected to be a new tool for sensitive assays and efficient therapeutics of diseases.However,due to the limitation in the understanding of the structure of aptamer-target complexes,research on covalent aptamers is not yet extensively promoted,and the application of covalent aptamers is still in the initial stage.Therefore,a systematic study on the performance and application of covalent aptamers is beneficial to further promote the performance improvement and practical application of aptamers.In this thesis,a proximity-driven covalent crosslinking strategy was developed to construct three types of covalent aptamers with different covalent label modifications.The performance of covalent aptamers was analyzed,and the application of NHS-modified covalent aptamers in molecular diagnosis,targeted therapy,and tumor imaging were explored in depth.Firstly,three types of covalent aptamers modified with different covalent labels were constructed based on the proximity-driven covalent cross-linking strategy.The cross-linking reaction efficiency,binding rate,and cross-linking specificity of the three types of covalent aptamers to the target were analyzed.Through a systematic comparison of performance and from a comprehensive consideration of the reactivity and selectivity of covalent cross-linking,among the three types of covalent labels,the N-hydroxysuccinimide(NHS)modification was the most suitable for the construction of covalent aptamers to achieve the best balance of reactivity and selectivity.It can improve the stability of aptamers at the overall level.To investigate the performance of NHS-modified covalent aptamers and their application in molecular diagnosis,the binding affinities of covalent aptamers,non-covalent aptamers,and commercial antibodies were systematically compared using the surface plasmon resonance technique.Covalent aptamers were found to have eminent binding affinity to the targets.The ability of covalent aptamers to resist various environmental stresses was further verified by combining direct enzyme-linked immunosorbent assay(ELISA)and denaturing agarose gel electrophoresis.After that,a sandwich ELISA was constructed based on the covalent aptamer to achieve an ultrasensitive detection of severe acute respiratory syndrome coronavirus 2(SARSCoV-2)nucleocapsid protein.Finally,the cyclic amplification ELISA was developed by combining the rolling cyclic amplification reaction and covalent aptamers,demonstrating the critical application of covalent aptamers in the molecular diagnosis of biomarkers.To further investigate the application of NHS-modified covalent aptamers in targeted therapy,a covalent aptamer was developed as a neutralizing reagent for blocking SARS-CoV-2 infection.The abilities of covalent neutralizing aptamer,non-covalent neutralizing aptamer,and neutralizing antibody to target SARS-CoV-2 were systematically compared.It was found that covalent neutralizing aptamer could efficiently and stably bind to SARSCoV-2.The effect of covalent binding on the stability of aptamer-target complexes was further assessed using stability analysis techniques.The effective blockade of SARS-CoV-2 infection by covalent neutralizing aptamer was further validated by combining pseudovirus neutralization analysis and neutralization assays in a human blood circulation simulation system.Finally,to investigate the application of NHS-modified covalent aptamers in tumor imaging,a pH-driven targeting covalent nanoprobe was developed by utilizing the pH-responsive nucleic acid structure i-motif,truncated aptamers,and proximity-driven covalent cross-linking strategies.The covalent nanoprobe can specifically bind to cancer cells in the tumor microenvironment.Compared with conventional non-covalent aptamer,the pH-driven targeting covalent nanoprobe enables reactive recognition and durable binding of targets.Applying the pH-driven targeting covalent nanoprobe to tumor imaging,it was found that the covalent binding of covalent aptamers to targets can avoid binding-then-shedding of the probe and enable long-term tumor imaging in the tumor microenvironment.In summary,this thesis presents a relatively comprehensive performance analysis and application study of covalent aptamers.This work is an important guide for the in-depth investigation of the molecular functions and application performance of covalent aptamers.