Selection And Identification Of DNA Aptamer Against Human Bladder Cancer

Objective:To explore the value of CELL-SELEX(CELL based Systematic Evolution of Ligands by Exponential enrichment) to screen aptamers against human bladder cancer cell(EJ cell). By detectiing aptamer affinity and specificity against EJ cells, we evaluate whether aptamers could be applied to diagnose bladder urothelial carcinoma (Bladder Urothelial Carcinoma, BUC) as a molecular probe.Methods:1. A single-stranded DNA (ssDNA) library was constructed using chemical synthesis method in vitro. EJ cells were used as positive screening cells and immortalized cells derived from normal human urothelial cells (HCV29) as negative screening cells.2. ssDNA fragments obtained from each round were amplified by PCR, then the length of PCR products were detected by agarose gel electrophoresis. The screening process was monitored by flow-cytometry.3. According to the results of flow cytometry, ssDNA pool with the highest binding rate(BR) was amplified by PCR. PCR products were purified using DNA purification Kit, then cloned and sequenced. According to sequencing results, the secondary structure of ssDNA was analysed and predicted with RNA structure software.4. The fluorescence intensity of aptamers binding EJ cells was detected by flow cytometry. According to the result of flow cytometry, Kd of aptamer was calculated to analyze aptamer affinity against target cells.5. The aptamers with the highest affinity were marked with FITC fluorophore. The aptamers were incubated with EJ cells, HCV29 cells, HepG2 cells and 293T cells respectively. Binding force between aptamer and above cells was measured by flow cytometry to analyze aptamer specificity against target cells.Results:1. The primary library of DNA was constructed with ssDNA. The total length of ssDNA is 91nt. Both ends of ssDNA were fixed sequence with the lengh of 21nt, and the middle sequence with the length of 49nt was random. After 13 rounds of EJ cells screening, ssDNA against EJ cells were selected and the length of DNA fragment was about 91bp detected by agarose gel electrophoresis.2. The result of sequencing showed that ssDNA contained three different kinds of nucleotide sequence, named L1, L2, L3 respectively. The prediction of three ssDNA secondary structure were maily stem-loop structure. The structural characteristics of three ssDNA was consistent with that of aptamers.3. Three kind of aptamers binded EJ cells respectively and Kd of aptamer L1 was 35.2nM, lower than those of aptamer L2(Kd=82.7nM), L3(Kd=93.5nM). Binding force between aptamers L1 and EJ cells was the highest than that of aptamer L2, L3. The result of flow cytometry showed that aptamer L1 targeted to bind EJ cell specifically, but no binding action was found between L1 and HCV29、293T or HepG2.Conclusions:We generated aptamer L1 against human bladder cancer cell(EJ cell) by the CELL-SELEX technology. The secondary structure of LI was consistent with that of aptamers. And aptamer L1 selectively binded EJ cells with high affinity.