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Abstract Aptamers are nucleic acid bioreceptors that have been used in various applications including medical diagnostics and as therapeutic agents. Identifying the most optimal aptamer for a particular application is very challenging. Here, we for the first time have developed a high-throughput method for accurately quantifying aptamer binding affinity, specificity, and cross-reactivity via the kinetics of aptamer digestion by exonucleases. We demonstrate the utility of this approach by isolating a set of new aptamers for fentanyl and its analogs, and then characterizing the binding properties of 655 aptamer–ligand pairs using our exonuclease digestion assay and validating the results with gold-standard methodologies. These data were used to select optimal aptamers for the development of new sensors that detect fentanyl and its analogs in different analytical contexts. Our approach dramatically accelerates the aptamer characterization process and streamlines sensor development, and if coupled with robotics, could enable high-throughput quantitative analysis of thousands of aptamer–ligand pairs.
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High-throughput methods in aptamer discovery and analysis
Aptamers are small, functional nucleic acids that bind a variety of targets, often with high specificity and affinity. Genomic aptamers constitute the ligand-binding domains of riboswitches, whereas synthetic aptamers find applications as diagnostic and therapeutic tools, and as ligand-binding domains of regulatory RNAs in synthetic biology. Discovery and characterization of aptamers has been limited by a lack of high-throughput approaches that uncover the target-binding domains and the biochemical properties of individual sequences. With the advent of high-throughput sequencing, large-scale analysis of in vitro selected populations of aptamers (and catalytic nucleic acids, such as ribozymes and DNAzmes) became possible. In recent years the development of new experimental approaches and software tools has led to significant streamlining of the selection-pool analysis. This article provides an overview of post-selection data analysis and describes high-throughput methods that facilitate rapid discovery and biochemical characterization of aptamers.
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- Award ID(s):
- 1804220
- PAR ID:
- 10273352
- Date Published:
- Journal Name:
- Methods in enzymology
- Volume:
- 621
- ISSN:
- 0076-6879
- Page Range / eLocation ID:
- 329-346
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1016/bs.mie.2019.02.009
