Study On Selection Of Human PDL1 Aptamer

Background:Conventional tumor diagnosis methods have low sensitivity and poor specificity,and tumors are likely to be detected in the middle and late stages,so that tumors cannot be treated in time.In addition,the traditional methods of tumor treatment have a large side effect,and the therapeutic effect of the drug is not high and may even have toxic effects on normal tissues.Therefore,the diagnosis and treatment of tumors still face enormous challenges.In recent years,immunological checkpoint blockade has produced enormous clinical application potential in cancer treatment.The programmed cell death-1 receptor(PD1)is expressed on T cells and interacts with the homologous ligand-programmed death ligand 1(PDL1,also known as CD274)on tumor cells and is one of the regulatory pathways that control the immune response.The study of this PD1/PDL1 check site helps us to study the mechanism of tumor immune escape and control the development of tumors to kill tumors.Aptamers are a type of DNA or RNA single oligonucleotide molecule derived from a random DNA or RNA oligonucleotide chain by systematic evolution of ligands by exponential enrichment(SELEX).The results were screened in the library.The obtained nucleic acid aptamer is capable of specifically binding different targeting molecules by its own specific three-dimensional structure.A nucleic acid aptamer is functionally similar to an antibody and,therefore,is also referred to as a"chemical antibody".Due to their high affinity and specificity and low immunogenicity,nucleic acid aptamers are a hot topic in current cancer research.Clustered regularly interspaced short palindromic repeat/CRISPR associated protein 9(CRISPR/Cas9)is a powerful gene editing tool with unlimited potential in biomedical research.We first constructed a cell line that stably and highly expressed human PDL1(h PDL1)protein using CRISPR/Cas9technology,and then used Cell-SELEX technology to screen for a nucleic acid aptamer that specifically recognizes human PDL1 protein.Screening for PDL1aptamers can help us study the immune escape mechanisms of tumors and develop targeted drugs that are beneficial for tumor therapy.Objective:The high-affinity and high-specificity nucleic acid aptamer sequences of human PDL1 protein were screened in vitro by Cell-SELEX technology,and their biological functions were studied,which provided new ideas for anti-tumor research.Methods:The human PDL1 protein was integrated into the h AAVS1 safety site by CRISPR/Cas9 technology,and a cell line stably expressing human PDL1protein(293T-h PDL1)was constructed.The expression of human PDL1 protein in the cell line was detected by flow cytometry.The cell line was used as a positive screening cell,while 293T was used as a negative screening cell.A DNA oligonucleotide library consisting of 81 bases fixed at both ends and random in the middle was synthesized and then screened by Cell-SELEX technique.The screening pressure was continuously increased during the screening process,and the enrichment of the screening products was detected by flow cytometry.When the enrichment reached saturation,the screening was stopped and the final product was subjected to TA cloning.Sequence alignment analysis and secondary structure investigation were performed on the sequencing results,and sequences with higher frequency of repeated sequences were selected as candidate aptamers.Flow cytometry was used to detect the affinity of each candidate aptamer,and the equilibrium dissociation constant(K_d value)was obtained,and specific detection was performed.Select a candidate aptamer with high specificity and strong affinity for sequence optimization truncation,and use flow cytometry and confocal microscope imaging to specifically detect it.Results:1.This study used Cell-SELEX technology to successfully obtain 6nucleic acid aptamers that specifically bind to target cell 293T-h PDL1 after 18rounds of screening,h PDL1-1,h PDL1-2,h PDL1-3,h PDL1-4,h PDL1-5 and h PDL1-6.2.The K_d values of these 6 candidate aptamers are all nanomolar range,and according to the detection of binding ability,h PDL1-6 is the best aptamer.The K_d value is 74.01±12.68 n M,which has good targeting effect.3.At 4℃and37℃ambient temperature,there is no obvious difference in the binding capacity of h PDL1-6.4.Truncate the sequence of h PDL1-6 to obtain three aptamers,h PDL1-6a,h PDL1-6b and h PDL1-6c.The results show that h PDL1-6b has the best binding ability.Conclusions:The CRISPR/cas9 technology was able to construct a cell line stably expressing human PDL1 protein(293T-h PDL1)for screening human PDL1nucleic acid aptamers.This study successfully screened the nucleic acid aptamer h PDL1-6b that binds human PDL1 protein with high affinity and high specificity,which provides a new idea for anti-tumor research.