期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 107, 期 35, 页码 15373-15378出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1009331107
关键词
high-throughput DNA sequencing; PDGF-BB; Quantitative Selection of Aptamers through Sequencing; MicroMagnetic Separation device
资金
- Army Research Office (ARO) Institute for Collaborative Biotechnologies
- Office of Naval Research
- Charlotte Geyer Foundation
- Midwest Progenitor Cell Consortium
- National Institutes of Health
- Korea Research Foundation [KRF-2008-357-C00086]
- National Research Foundation of Korea [2008-357-C00086] Funding Source: Korea Institute of Science & Technology Information (KISTI), National Science & Technology Information Service (NTIS)
We describe the integration of microfluidic selection with high-throughput DNA sequencing technology for rapid and efficient discovery of nucleic acid aptamers. The Quantitative Selection of Aptamers through Sequencing method tracks the copy number and enrichment-fold of more than 10 million individual sequences through multiple selection rounds, enabling the identification of high-affinity aptamers without the need for the pool to fully converge to a small number of sequences. Importantly, this method allows the discrimination of sequences that arise from experimental biases rather than true high-affinity target binding. As a demonstration, we have identified aptamers that specifically bind to PDGF-BB protein with K(d) < 3 nM within 3 rounds. Furthermore, we show that the aptamers identified by Quantitative Selection of Aptamers through Sequencing have similar to 3-8-fold higher affinity and similar to 2-4-fold higher specificity relative to those discovered through conventional cloning methods. Given that many biocombinatorial libraries are encoded with nucleic acids, we extrapolate that our method may be extended to other types of libraries for a range of molecular functions.
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