Discovering aptamers by cell-SELEX against human soluble growth factors ectopically expressed on yeast cell surface

Hsien Wei Meng, John M. Pagano, Brian S. White, Yoshiko Toyoda, Irene M. Min, Harold G. Craighead, David Shalloway, John T. Lis, Kai Xiao, Moonsoo M. Jin

Research output: Contribution to journalArticle

2 Scopus citations

Abstract

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, we sought to discover DNA aptamers against human serum markers for potential therapeutic and diagnostic applications. To circumvent soluble expression and immobilization for performing SELEX, we 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. 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 anti-VEGF antibody drug, bevacizumab. Considering the fact that many mammalian secretory proteins have been functionally expressed in yeast, the strategy of implementing cell-SELEX and quantitative binding assay can be extended to discover aptamers against a broad array of soluble antigens.

Original languageEnglish (US)
Article numbere93052
JournalPLoS ONE
Volume9
Issue number3
DOIs
StatePublished - Mar 27 2014

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)
  • General

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