Screening And Characterization Of Aptamers In The Serum Of Gastric Cancer By Bidirectional Thermal Circulation Subtractive SELEX Technology

Gastric cancer is one of the most common malignant tumors with high incidence in China,seriously threatening the health of human.Due to the lacking of a mature examination system and obvious symptoms of early gastric cancer,it was too late when founded out.Improving the detection rate of early gastric cancer is of great significance for improving the treatment of gastric cancer.Detection of serum tumor markers are noninvasive and convenient,but there are also some problems such as low sensitivity and specificity,which seriously affect the clinical diagnosis and treatment of gastric cancer.Therefore,screening high affinity and specificity nucleic acid aptamers in the serum of gastric cancer is the key point.In this study,bidirectional thermal circulation subtractive SELEX technology were technical support,and agar-magnetic beads were used as carriers,gastric cancer serum and non-cancer serum were both as positive and negatice screening targets,then a quick and efficient serum detection technology was established.After that,the affinity and specificity of the aptamers were detected by ELISA method.At last,the sequence and the targeted protein information obtained by high-throughput sequencing and Label free were studied with the help of bioinformatics software.The main research contents and results are as follows:1)Target protein-coated magnetic beads were first incubated with the initial ssDNA library for 5 rounds to fully enrich the specific ssDNA,and then subtractive SELEX screening techonlogy was added from the 6th round.At the same time,the screening conditions were optimized to increase the specificity of aptamers.After 19 rounds of bidirectional screening,gastric cancer and non-cancer serum specific aptamers were obtained from the random ssDNA library and then high-throughput sequencing was performed.The result shows that ten gastric cancer serum specific and nine non-cancer serum specific aptamer sequences were with a richness of 1%,these sequences were then numbered and synthesized to analysis.2)The affinity and specificity of aptamers with a richness of 10% were verified by ELISA method,and the results shows that the affinity between the aptamer sequences with high abundance and the target protein was at the nanomolar level.And the aptamer named G-AP2,G-AP3,N-AP1 and N-AP3 have small dissociation constants,showing a strong affinity.After that,high-abundance aptamer was used to establish a bidirectional detection technology to verify the 100 gastric cancer serum and non-cancer serum cases.Statistical analysis result shows that the positive detection rate of serum is 90%.3)Online sequence analysis software NUPACK was used to analyze the secondary structure of the aptamer and DNAMAN was used for sequence alignment.The results shows that the sequence of aptamers were mostly stem loop shaped and rich in base-paired rigions.4)The target proteins of aptamers specific to gastric cancer or non-cancer serum were extracted by biotin-avidin assay.The Label free technique was used for protein spectrum analysis and the online analysis software omicsbean was used to the functional annotation and pathway analysis of aptamer specific proteins of gastric cancer serum.The results shows that 145 target proteins were mainly located in the extracellular region and could bind with cell surface receptors to participate in the transport of intracellular and extracellular substances.These proteins are also involved in gene transcription,cell migration and apoptosis,protein sorting and some tumorigenesis related signaling pathways,which shows the potential of aptamer as a tumor marker in clinical detection and treatment of cancer.In conclusion,bidirectional thermal circulation subtractive SELEX technology can accurately screening the gastric cancer or non-cancer serum specific nucleic acid aptamer.Gastric cancer and non-cancer serum could be accurately determined by real-time quantitative PCR detection.Nucleic acid and protein sequence information were analyzed by high-throughput sequencing and mass spectrometry,which provide a new reference for the early clinical diagnosis and treatment of gastric cancer.