Selection Of Aptamers Against Human Glioma Cell SHG44 And Preliminary Application

Objective: Using the technology of Cell-SELEX to obtain aptamers with high specificity and affinity against human glioma cell SHG44 but not for human normal astroglia cell SVGp12.Methods: The single-stranded oligonucleotide library with a length of 80 bases and a number of about 1013-1014 was synthesized by DNA synthesizer.The 5'end and the 3'end of the nucleotide library are known as complementary sequences of positive and negative primers,and the 40 bases in the middle are random sequences in the DNA synthesis process.The synthetic single-stranded DNA library was then incubated sufficient with human normal astroglia cell SVGp12 and collected nucleic acid sequences that did not bind to the surface of human normal astroglia cell SVGp12.The collected sequences were then incubated again with human glioma cell SHG44 to isolate those nucleic acid sequences that bind to the surface of human glioma cell SHG44.These single-stranded nucleic acids were subjected to PCR amplification and enrichment for the next screening of the DNA library,so the binding force of the human glioma cell SHG44 with the second round of DNA library is stronger than the initial round,and the the binding capacity of human normal astroglia cell SVGp 12 is getting weaker.Therefore,after several rounds of repeated selecting and amplification,we achieve the nucleic acid library that the binding force reaching the saturation state against human glioma cell SHG44.By sequencing 70 sequences of the last round of DNA library,those DNA sequences with high repetition rate were selected for batch synthesis and characterization.We select the single DNA sequence with the strongest binding ability to the target cell and the most stable structure to optimize,and the shortest aptamer was obtained.Finally,the affinity,specificity,properties of the cell-binding target and the stability of the physiological temperature were investigated by flow cytometry and fluorescence confocal microscopy.Results: 1.Aptamer S6-1-B with highly specific and high affinity binding to human glioma cell SHG44 was obtained by 10-round screening,optimization and characterization using Cell-SELEX technique.2.The length of the aptamer S6-1-B was 53 bases.The secondary structure was stable,and the binding ability to human glioma cell SHG44 was 8-12 times higher than the original library.3.This aptamer still retain the high specificity and high affinity for SHG44 under the physiological temperature of 37?.4.The aptamer S6-1-B binds to the cell membrane of the target cell SHG44,and its binding target is a proteinaceous substance.5.FAM and HS-SH-T-Spacer9-Biotin-modified aptamer S6-1-B retains good specificity and high affinity for target cells,and the modified aptamer S6-1-B is used to initially identify membrane surface biomarker of human glioma cell SHG44.6.A preliminary targeted drug therapy experiment was performed on human glioma cell SHG44 by S6-1-B carrying adriamycin.Conclusion: We successfully screened the specific aptamer S6-1-B obtained from human glioma cell SHG44.The aptamer S6-1-B was modified to be used in the biomarker sequencing and targeted drug therapy.This study further improved the genealogy of the specific aptamers of various glioma cell s.The aptamer S6-1-B can be used as a probe for cell imaging of brain gliomas,targeted drug delivery,biomarker discovery,and tumor prognosis with the high specificity,high affinity,and binding to the target cell surface protein.