title: Discovering aptamers by cell-SELEX against human soluble growth factors ectopically expressed on yeast cell surface.
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.
authors.affiliation: Department of Biomedical Engineering, Cornell University, Ithaca, New York, United States of America; Department of Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, New York, United States of America.
authors.forename: Hsien-Wei
authors.initials: HW
authors.lastname: Meng
authors.affiliation: Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York, United States of America.
authors.forename: John M
authors.initials: JM
authors.lastname: Pagano
authors.affiliation: Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York, United States of America.
authors.forename: Brian S
authors.initials: BS
authors.lastname: White
authors.affiliation: Department of Biomedical Engineering, Cornell University, Ithaca, New York, United States of America.
authors.forename: Yoshiko
authors.initials: Y
authors.lastname: Toyoda
authors.affiliation: Department of Biomedical Engineering, Dongguk University, Seoul, South Korea.
authors.forename: Irene M
authors.initials: IM
authors.lastname: Min
authors.affiliation: School of Applied and Engineering Physics, Cornell University, Ithaca, New York, United States of America.
authors.forename: Harold G
authors.initials: HG
authors.lastname: Craighead
authors.affiliation: Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York, United States of America.
authors.forename: David
authors.initials: D
authors.lastname: Shalloway
authors.affiliation: Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York, United States of America.
authors.forename: John T
authors.initials: JT
authors.lastname: Lis
authors.affiliation: Department of Radiology, Weill Cornell Medical College, New York, New York, United States of America.
authors.forename: Kai
authors.initials: K
authors.lastname: Xiao
authors.affiliation: Department of Biomedical Engineering, Cornell University, Ithaca, New York, United States of America; Department of Radiology, Weill Cornell Medical College, New York, New York, United States of America; Department of Biomedical Engineering, Dongguk University, Seoul, South Korea.
authors.forename: Moonsoo M
authors.initials: MM
authors.lastname: Jin
mesh_terms: D052157:Aptamers, Nucleotide / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D001483:Base Sequence; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D061505:Cell Surface Display Techniques; D016384:Consensus Sequence; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018919:Neovascularization, Physiologic / Q000187:drug effects / Q000235:genetics; D009690:Nucleic Acid Conformation; D059372:Nucleotide Motifs; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D052156:SELEX Aptamer Technique; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism; D015003:Yeasts / Q000235:genetics / Q000378:metabolism*
chemical_list: D052157:Aptamers, Nucleotide; D036341:Intercellular Signaling Peptides and Proteins; D042461:Vascular Endothelial Growth Factor A