Discovering aptamers by cell-SELEX against human soluble growth factors ectopically expressed on yeast cell surface. Academic Article uri icon

Overview

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 (K(D) = ∼ 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.

publication date

  • March 27, 2014

Research

keywords

  • Aptamers, Nucleotide
  • Cell Membrane
  • Intercellular Signaling Peptides and Proteins
  • SELEX Aptamer Technique
  • Yeasts

Identity

PubMed Central ID

  • PMC3968096

Scopus Document Identifier

  • 84899854641

Digital Object Identifier (DOI)

  • 10.1371/journal.pone.0093052

PubMed ID

  • 24675636

Additional Document Info

volume

  • 9

issue

  • 3