Development Of Dna Aptamers By Cell-Selex Using Yeast Cell Surface Display
Files
No Access Until
Permanent Link(s)
Collections
Other Titles
Author(s)
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, 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.
Journal / Series
Volume & Issue
Description
Sponsorship
Date Issued
Publisher
Keywords
Location
Effective Date
Expiration Date
Sector
Employer
Union
Union Local
NAICS
Number of Workers
Committee Chair
Committee Co-Chair
Committee Member
Lis, John T
Sondermann, Holger