In Vitro Selection And Identification Of Aptamers Against Human Lung Adenocarcinoma Cell A549

Objective To develop a cell-SELEX method and screen aptamers with high affinity and specificity against lung adenocarcinoma cell A549. The aptamers selected from cell-SELEX may be further used to distinguish cancer and normal cells.Methods An ssDNA library containing randomized 35-base nucleotides and primers were synthesized chemically. The library was first incubated with the target cells. After washing, DNA molecules bound to the target cell surface were collected and amplified for the next round of selection. The ssDNA library for the next round were prepared by passing through Streptavidin Sepharose columns and denatured in NaOH before incubation with the target cell. Selection pressure was adjusted by shortening the incubation time, increasing the washing times and using a longer wash time. Negative selection against Beas-2b was performed after the positive selection in round 4 and 12, and all DNA sequences that bound Beas-2b were removed. Flow cytometry was used to monitor the affinity of the ssDNA binding to the target cell. Enriched pools of round 15 and 20 were cloned, sequenced and analyzed. Finally, the second structure of aptamers identified in round 15 and 20 was predicted and analyzed by RNA Structure software.Results The mean fluorescence index of unselected pool and 20th pool against A549 cell was 0.544 and 1.73, respectively. The positive rate for fluoresence was 0.1% and 4.7%, which was significantly increased, while this was not observed in unselected, 16th, 20th pools in comparison to control cell Beas-2b. Two pairs of the aptamers with the same sequences were found in selection rounds 15 and 20, respectively. According to the characteristics of the second structures, the aptamers can be divided into three families, namely families 1, 2 and 3. The families 1, 2 and 3 consisted of 40.8%, 32.7% and 14.3% of the aptamers in round 15,respectively. And the families 1, 2 and 3 consisted of 46%, 4%, 32% in round 20, respectively.Conclusions We developed a cell-SELEX method and generated a group of aptamers against human lung adenocarcinoma cancer cell A549. These aptamers may be used for detection and identification of lung cancer cells.