Identification and Characterization of an eIF4e DNA Aptamer That Inhibits Proliferation With High Throughput Sequencing

Molecular Therapy: Nucleic Acids

Kiat Whye Kong¹  Wei Mei Guo¹  Shawn Hoon¹  Yiqi Seow¹  Hui Ling Yeo¹  Christopher John Brown²  Soo Tng Quah² 

[1] Molecular Engineering Laboratory, Biomedical Sciences Institutes, Agency for Science Technology and Research, Singapore;p53 Laboratory, Biomedical Sciences Institutes, Agency for Science Technology and Research, Singapore;

关键词: Aptamer;    eIF4e;    SELEX;    proliferation;   

DOI  :  10.1038/mtna.2014.70

来源: DOAJ

【 摘 要 】

Development of DNA aptamer screens that are both simple and informative can increase the success rate of DNA aptamer selection and induce greater adoption. High eIF4e levels contribute to malignancies, thus eIF4e presents itself as a valuable target for DNA aptamer-based inhibition screen. Here, we demonstrate a method for the rapid selection of looped DNA aptamers against eIF4e by combining negative selection and purification in a single step, followed by characterization with high throughput sequencing. The resulting aptamers show functional binding to eIF4e and inhibit translation initiation in biochemical assays. When transfected into cells, eIF4e aptamers cause a dramatic loss of cell proliferation in tumor cells as seen with eIF4e knockdown with antisense oligonucleotides, shRNAs, and siRNAs, hinting at therapeutic possibilities. With the large data set provided by high throughput sequencing, we demonstrate that selection happens in waves and that sequencing data can be used to infer aptamer structure. Lastly, we show that ligation of looped aptamers can enhance their functional effects. These results demonstrate a rapid protocol to screen and optimize aptamers against macromolecules of interest.

【 授权许可】

Unknown