An in solution assay for interrogation of affinity and rational minimer design for small molecule-binding aptamers

The Analyst
Nadine R FrostMaria C DeRosa

Abstract

Aptamers are short single-stranded oligonucleotides that fold into unique three-dimensional structures, facilitating selective and high affinity binding to their cognate targets. It is not well understood how aptamer-target interactions affect regions of structure in an aptamer, particularly for small molecule targets where binding is often not accompanied by a dramatic change in structure. The DNase I footprinting assay is a classical molecular biology technique for studying DNA-protein interactions. The simplest application of this method permits identification of protein binding where DNase I digestion is inhibited. Here, we describe a novel variation of the classical DNase I assay to study aptamer-small molecule interactions. Given that DNase I preferentially cleaves duplex DNA over single-stranded DNA, we are able to identify regions of aptamer structure that are affected by small molecule target binding. Importantly, our method allows us to quantify these subtle effects, providing an in solution measurement of aptamer-target affinity. We applied this method to study aptamers that bind to the mycotoxin fumonisin B1, allowing the first identification of high affinity putative minimers for this important food contaminant. We...Continue Reading

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Citations

Jul 21, 2015·Analytical Chemistry·Maureen McKeagueMaria C DeRosa
Jun 1, 2016·Frontiers in Chemistry·Annamaria Ruscito, Maria C DeRosa
Sep 28, 2016·The Journal of Steroid Biochemistry and Molecular Biology·Markéta SvobodováCiara K O'Sullivan
Dec 15, 2017·Current Protocols in Chemical Biology·Annamaria RuscitoMaria C DeRosa
Feb 9, 2020·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Cao-An Vu, Wen-Yih Chen
Jan 13, 2021·Biosensors·Vicente Antonio Mirón-MéridaFrancisco M Goycoolea
Feb 19, 2021·Journal of Agricultural and Food Chemistry·Emily MastronardiMaria C DeRosa
Apr 26, 2021·Analytica Chimica Acta·Vicente Antonio Mirón-MéridaFrancisco M Goycoolea

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