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Title: Label-free profiling of DNA aptamer-small molecule binding using T5 exonuclease
Abstract In vitro aptamer isolation methods can yield hundreds of potential candidates, but selecting the optimal aptamer for a given application is challenging and laborious. Existing aptamer characterization methods either entail low-throughput analysis with sophisticated instrumentation, or offer the potential for higher throughput at the cost of providing a relatively increased risk of false-positive or -negative results. Here, we describe a novel method for accurately and sensitively evaluating the binding between DNA aptamers and small-molecule ligands in a high-throughput format without any aptamer engineering or labeling requirements. This approach is based on our new finding that ligand binding inhibits aptamer digestion by T5 exonuclease, where the extent of this inhibition correlates closely with the strength of aptamer-ligand binding. Our assay enables accurate and efficient screening of the ligand-binding profiles of individual aptamers, as well as the identification of the best target binders from a batch of aptamer candidates, independent of the ligands in question or the aptamer sequence and structure. We demonstrate the general applicability of this assay with a total of 106 aptamer-ligand pairs and validate these results with a gold-standard method. We expect that our assay can be readily expanded to characterize small-molecule-binding aptamers in an automated, high-throughput fashion.  more » « less
Award ID(s):
1905143 2135005
PAR ID:
10199511
Author(s) / Creator(s):
; ; ; ;
Date Published:
Journal Name:
Nucleic Acids Research
Volume:
48
Issue:
20
ISSN:
0305-1048
Page Range / eLocation ID:
e120 to e120
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
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