Development Of Dna Aptamers By Cell-Selex Using Yeast Cell Surface Display
SELEX, the process of selecting aptamers, is often hampered by the difficulty of preparing target molecules in their native forms and by a lack of a simple yet quantitative assay for monitoring enrichment and affinity of reactive aptamers. In this study, I established a new method to seek to discover DNA aptamers against human serum markers for potential therapeutic and diagnostic applications. To circumvent soluble expression and immobilization for performing SELEX, I ectopically expressed soluble growth factors on the surface of yeast cells to enable cell-SELEX and devised a flow cytometry-based method to quantitatively monitor progressive enrichment of specific aptamers. High-throughput sequencing of selected pools revealed that the emergence of highly enriched sequences concurred with the increase in the percentage of reactive aptamers shown by flow cytometry. I first tested if the yeast-surface display works as a platform for examining bindings of aptamers to target proteins. Afterwards, I particularly selected DNA aptamers against VEGF were specific and of high affinity (KD = ~ 1 nM), and demonstrated a potent inhibition of capillary tube formation of endothelial cells, comparable to the effect of a clinically approved antiVEGF antibody drug, bevacizumab. I also have successfully selected DNA aptamers i against PDGF-A, PDGF-B. Considering the fact that many mammalian secretory proteins have been functionally expressed in yeast, the strategy of implementing cellSELEX and quantitative binding assay can be extended to discover aptamers against a broad array of soluble antigens.
Aptamer; Cell-SELEX; Yeast Surface Display
Coonrod, Scott A.; Lis, John T; Sondermann, Holger
Ph.D. of Veterinary Medicine
Doctor of Philosophy
dissertation or thesis