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

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

In Vitro Selection and Characterization of DNA Aptamers to a Small Molecule Target

“…Further details and strategies can be found in Frost et al(2015). By interpreting the apparent local K D s of binding between the full-length and minimal aptamer sequences, one can understand "binding regions" better.…”

“…Changes in the intensity of band patterns (cleaved products) can be used to determine the affinity. (Frost et al, 2015) Equilibrium dialysis The aptamer and a range of target concentrations are equilibrated in a two compartment cell separated by a semipermeable membrane that allows only the small molecule to pass through. The concentration of the small molecule in each compartment can be measured (e.g., using HPLC, fluorescence, MS) to determine the binding affinity.…”

“…Thus, the high affinity and specificity for certain targets may be achieved only with complex structures requiring long sequences (Luo et al, 2010;Nieuwlandt, 2000). The second challenge is that DNA is chemically stable; thus, chemically based cleavage probing to map boundary sites that is applied to RNA aptamers (Mannironi, Scerch, Fruscoloni, & Tocchini-Valentini, 2000) cannot be directly reproduced with DNA aptamers (Frost, McKeague, Falcioni, & DeRosa, 2015). A final challenge is that there is a lack of accurate structure prediction programs.…”

“…By interpreting the apparent local K D s of binding between the full-length and minimal aptamer sequences, one can understand "binding regions" better. Further details and strategies can be found in Frost et al(2015).…”

Gold Nanoparticles as a Platform for Small Molecule SELEX

“…FB139 is an aptamer previously selected for another small molecule mycotoxin, Fumonisin B1 (FB1). 19,49 This sequence is approximately 3 times the length compared to A08m. Using the same method, there was no consistent or significant difference in the amount of adsorption between these two sequences (Figure 2.15).…”