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.

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.

A strategy for aptamer-based biosensing termed linkage inversion assembled nano-aptasensors (LIANAs) is shown to be a generally applicable approach to the sensitive and specific detection of a target molecule in turn-on fluorescence solution-based and paper-based tests.

Fumonisin B1 (FB1) and ochratoxin A (OTA) are agriculturally important mycotoxins that have implications in human and animal health. Aptamers are short single-stranded oligonucleotides that can be used as molecular recognition elements for the sensitive and selective detection of mycotoxins. Aptamers for FB1 and OTA were screened using a novel DNase I footprinting assay to characterize their structure and insolution affinity. Two minimal binding aptamers for FB1 were identified that retain high binding affinity in solution and to a bound target. Progress was made towards fluorescence turn-on and turn-off sensors for FB1, utilizing semiconductor quantum dots and fluorescein in conjunction with metallic-and carbon-based quenchers. A fluorescence nano-aptasensor was developed as a simple paper test, with the capacity to detect OTA with a limit of detection between 10 -100 nM. Rapid, robust aptasensors for FB1 and OTA have promising applications for in-field screening of mycotoxin residues on crops.iii