Selection, Optimization And Application Of Aptamers For Cancer Cell

To develop molecular recognition probes with high specificity and sensitivity hassignificant value in early diagnosis of tumors, cancer metastasis alter and therapeuticeffect evaluation. The emergence of aptamers for cancer cell provide an idealmolecular recognition probe for cancer researches in the molecular level, and it alsobring a new opportunity for cancer researches. Currently, aptamers have been widelyused in the fields of biology, chemistry and medicine, and these become the hots potand keystone of the researches on bioanalytical chemistry. However, the existingnumber of aptamers are still limited, which seriously limits the extensive applicationof aptamers. Meanwhile, the steps of those methods for screening aptamer of cancercell are complicated and time-consuming. Therefore, it is very significant to establishthe fast and efficient screening method to achieve more effective molecular probes. Inaddition, it brings great promotion effect for the development of modern analytica lchemistry and molecular medicine to establish the novel and sensitive analyticalmethods, which integrate aptamers with the new technologies in physics, chemistryand materials. Based on these, the major goal of this thesis is to establish a highefficient screening method and technology, and then we use this method to achieve theaptamers bound to target with important value. Meanwhile, we integrate thenanotechnology and the biosensing technique to establish the aptamer sensor fortumor with high specificity and high sensitivity. Following several works have beenperformed.1. Selection and application of aptamers for human hepatocarcinoma cell lineHepG2cellsBased on the cell-SELEX technology, we focused on the factors and pressures ofthe aptamer selection. In order to improve the affinity and specificity, we successfullyoptimized the conditions and simplified the selection procedures, such as, the stabilityof the pool, the separation method, the buffer, ionic strength, the non-specificadsorption, the temperature and strength of incubation and washing. The selection ofhuman hepatocellular carcinoma (HCC) cell line HepG2specific aptamer wasperformed as a model, under this optimized strategy, we successfully obtained aaptamer recognized HepG2cells, further characterization shows that the aptamer havehigh affinity toward target cells with dissociation constant in nanomolar level and alldemonstrate high specificity against different cell lines. The excellent property o f this aptamer makes it a promising reagent in the construction of biochemical sensors andclinlical applications.2. The establishment and application of high efficient and SWCNT assistedcell-SELEX strategyBased on the traditional cell-SELEX strategy, we introduced the SWCNT into thecell-SELEX firstly, taking advantages of ssDNA adsorption ability of the SWCNT, wechoose the SWCNT as the unbound DNA remover, and changed the washing mode inthe traditional selection method. Using this strategy, we employed humannasopharyngeal carcinoma cell line CNE2as target, and HONE as control, thensuccessfully obtained an aptamer after6rounds of selection. Another selection usingtraditional cell-SELEX strategy without SWCNT, while it took15rounds to obtainaptamer with similar affinity. Therefore, the SWCNT assited cell-SELEX strategydemonstrated high efficiency in screening aptamers. The SWCNT can be used toremove the unwanted ssDNA that hardly bind to the target, and thus increase theseparation efficiency, it provide a new high efficient screening method and platformfor aptamer.3. Optimizing the sequence of acquired aptamerBased on the aptamers achieved by our laboratory, we optimized the structures ofthese sequences by analyzing the secondary structure of them. We analyzed theaptamers of many cancer cells, including nasopharyngeal carcinoma cell line CNE2cells, hepatocellular carcinoma cell line SMMC-7721cells and cholangiocarcinomacell line QBC-939. Taking aptamers GP6, T11, T12, zy1and yl19as example, basedon the data gathered by DNA/RNA structure analyzing software Mfold, we cut someof the sequence in the primer section, and changed some sequence to keep the stability.As a result, a serial of shorter aptamers GP47, T11a, T12a, ZYsls and yl19a wereachieved. And the length of these aptamer were cut from80to40nt (aptamer yl19aeven has23nt), the aptamers keep the similar stability, affinities and specificity, andthese aptamers provide simple and effective molecular recognition probes fordesigning and building biosensors. More importantly, these results will play animportant role in optimizing and designing of other aptamers.4. The establishment and application of aptasensor for sensitive detection ofcancer cells based on graphene oxideWe took advantage of the quenching capability of GO for dye-labelled aptamers,and aptamers were employed as the molecular recognition elements. As proof ofconcept, an aptamer sequence was selected using a cell-SELEX strategy in our laboratory for human liver cancer cell line SMMC-7721with high specificity and highaffinity, which was applied in this method. Samples with the target cells show highfluorescence intensity, while non-target samples still showed low fluorescenceintensity. This GO-based aptasensor exhibited high sensitivity and specificity with adetection limit of200SMMC-7721cells in200μL binding buffer. In addition, ourstrategy can directly detect target cells without washing and separation. Furthermore,the future design of detectors for other cancer cells only requires changing therecognition element for the target cell. More importantly, flow cytometry was used forthe specific recognition of target cells from a mixture of cells in fetal bovine serum todemonstrate the potential application of this method for medical diagnostics.5. The establishment and application of FRET-based split aptasensor for sensitivedetection of cancer cellsA pair of split aptamer probe was designed based on an aptamer screened by ourlab, and by combining it with FRET principle, we developed a FRET-based aptasensorfor sensitive detection of SMMC-7721cells. Only when in present of the target cells,the detectable signal generated by proximity of the dye labeled split probes. Thisaptasensor exhibited high sensitivity and specificity with a detection limit of20SMMC-7721cells in200μL binding buffer. We not only succeeded in the specificrecognition of target cells from a mixture of cells in20%fetal bovine serum, but alsoachieved to detect SMMC-7721cells in20%human serum, which demonstrated thisstrategy has great advantage in the detection of complex samples. In addition, ourstrategy can directly detect target cells without cell pretreatment, washing andseparation. We believe that the FRET-based split aptasensor shows great potential inbiochemical analysis and clinical applications.