Aptamer Selection For Human HCC Cell Line SMMC-7721and Sequence Design And Preliminary Application

The early diagnosis of cancer, accurate tumor typing and targeting therapy areamong the most challenging problems in biomedical field. Development of molecularprobes that can specifically recognize cancer cells is of great significance to realizethe early diagnosis of cancer, cancer metastasis warning and prognosis of cancertherapy. The recently developed cell-SELEX technology is able to abtain cell specificnucleic acid aptamers without previously knowing its target molecule. It provides anew strategy and idea for tumor specific biomarker discovery and cancer clinicaldiagnosis and typing, and it can offer important information for inlustratingtumoregenesis and development mechanisms on molecular level. In this work,cell-SELEX was adopted to achieve aptamers that can specifically recognize humanhepatocellular carcinoma (HCC) cells. Abtained aptamers were characterized andput into preliminary application.(1) Aptamer selectionCell-SELEX strategy was adopted with HCC cell line SMMC-7721as target cellsand HCC cell line HepG-2as control cells. The stringency of selection condition wasgradually increased to improve the screening efficiency. Only13rounds of selectionwere performed to reach the plateau, and the sequences are highly enriched in theselected pool. The most dominant four sequences can all bind with target cells.Further characterization shows that the four aptamers have great affinity toward targetcells with dissociation constant in nanomolar level and all demonstrate highspecificity against different cell lines. Moreover, they maintain their binding capacityat room temperature and body temperature.(2) Sequence analysis and designSequence evolution tendency among selected pools and the relationship betweensecondary structure of aptamers and their binding capacity were investigated.Refering to the information from homologous analysis and structure analysis, fivemore aptamers with excellent binding properties were hunt out from the13th roundselected pool. According to the aptamer (ZY1, ZY8, ZY11) structure information andthe experimental results of sequence truncation, we find that aptamer bind s with itstarget with a stem-loop-stem configuration. Sequence optimizition can be performedby shortening and strenthening its two stems while maintaining its binding capacity. Moreover, a split aptamer pair was designed based on the structure information. Thesplit aptamer pair can be induced by target to construct the stem-loop-stemconfiguration and generate fluorescence signal through FRET.(3) Preliminary application of aptamerIn order to investigate the aptamer target recogniton performance in complexbiological environment, the aptamer is applied to frozen tissue sections and in vivotumor imaging in mice. The experimental results show that the aptamer can stillmaintain good recognition ability in the complex environment with relatively hightissue specificity.