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

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 confirmed the binding affinity of these minimers using a magnetic bead binding assay.

Comprehensive analytical comparison of strategies used for small molecule aptamer evaluation.

Nucleic acid aptamers are versatile molecular recognition agents that bind to their targets with high selectivity and affinity. The past few years have seen a dramatic increase in aptamer development and interest for diagnostic and therapeutic applications. As the applications for aptamers expand, the need for a more standardized, stringent, and informative characterization and validation methodology increases. Here we performed a comprehensive analysis of a panel of conventional affinity binding assays using a suite of aptamers for the small molecule target ochratoxin A (OTA). Our results highlight inconsistency between conventional affinity assays and the need for multiple characterization strategies. To mitigate some of the challenges revealed in our head-to-head comparison of aptamer binding assays, we further developed and evaluated a set of novel strategies that facilitate efficient screening and characterization of aptamers in solution. Finally, we provide a workflow that permits rapid and robust screening, characterization, and functional verification of aptamers thus improving their development and integration into novel applications.