Applications Of Aptamers In Cancer Cell Recognition,Marker Discovery And Gene Editing Regulation

As a new type of molecular probe,aptamers have been widely used in biological analysis,detection and biomedicine due to their advantages of high affinity,good specificity,and wide target range.The research of this dissertation focused on the specific recognition and allosteric regulation by aptamers:1.Aptamer screening against primary colon cancer cell SW480We have successfully identified a DNA aptamer 151c for primary colon cancer cell SW480.Based on the whole-cell SELEX,primary cancer cell SW480 was used as targeted cell and cells from the same patient’s lymph node metastasis tissue as competitive agents.Under the high pressure SELEX condition,the identified aptamer 151c have good binding affinity and specificity to primary colon cancer-derived cell lines instead of metastasis cell lines.The aptamer was able to analyze mixed cell samples in physiological or bio-complex conditions and used for cancer cell imaging.151c showed lower binding capacity with trypsin treated cell indicating that 151c bound with a cell surface protein.Based on its excellent ability to distinguish the characteristics of metastasis and in situ cancer,this aptamer has potential application value in the study of cancer metastasis phenotype and mechanism.2.Aptamer AP-1 "fishing" tumor markers of non-small lung cancer A549 cell.The identification of tumor related cell membrane protein targets is important in understanding tumor progression,development of new diagnostic tools,and potentially for identifying new therapeutic targets.AP-1 aptamer was used as a fishhook to "bite" the potential biomarker of non-small cell lung cancer A549 cells.Biotin-avidin strategy was used to pull down aptamer-bound protein among A549 cell lysis.With the help of gel electrophoresis and mass spectrometry,nucleolin and nuclear heterogeneous heteroribonucleoprotein U were identified.They were related to the occurrence and development of tumors as report.Binding assay with protein-displayed beads and over-expressed cells were performed to confirm the relationship between AP-1 with these two proteins.The results both showed the aptamer had high affinity and selectivity with them.Additionally,we also found a higher nucleolin content in the serum of cancer patients than normal samples using ELISA analysis.This study demonstrates that nucleolin can be used as a broad-spectrum tumor marker for cancer diagnostic.3.Theophylline regulated Aptamer-based CRISPR/Cas 9 gene editingPrecise temporal and spatial control over genome editing agent such as CRISPR/Cas 9 would enable more selective and safer to manipulate the human genome.Benefited from ligand binding induced conformational changes,aptamers are useful tools for allosteric regulation.Here we designed a series of theophylline aptamer inserted sgRNA sequences to develop a universal small molecule regulated gene editing system.The aptamer sequence was inserted into the scaffold of sgRNA,which is irrelevant to target-specific sequence.Compared with current methods relying on guide RNA relevant operation,our allosteric sgRNA enables universal regulation of CRISPR/Cas9 systems simply by editing the corresponding guide RNA as usual,endowing the proposed system with broad application prospects in gene editing and therapy.