Engineering a Structure Switching Mechanism into a Steroid-Binding Aptamer and Hydrodynamic Analysis of the Ligand Binding Mechanism

Abstract

The steroid binding mechanism of a DNA aptamer was studied using isothermal titration calorimetry (ITC), NMR spectroscopy, quasi-elastic light scattering (QELS), and small-angle X-ray spectroscopy (SAXS). Binding affinity determination of a series of steroid-binding aptamers derived from a parent cocaine-binding aptamer demonstrates that substituting a GA base pair with a GC base pair governs the switch in binding specificity from cocaine to the steroid deoxycholic acid (DCA). Binding of DCA to all aptamers is an enthalpically driven process with an unfavorable binding entropy. We engineered into the steroid-binding aptamer a ligand-induced folding mechanism by shortening the terminal stem by two base pairs. NMR methods were used to demonstrate that there is a transition from a state where base pairs are formed in one stem of the free aptamer, to where three stems are formed in the DCA-bound aptamer. The ability to generate a ligand-induced folding mechanism into a DNA aptamer architecture based on the three-way junction of the cocaine-binding aptamer opens the door to obtaining a series of aptamers all with ligand-induced folding mechanisms but triggered by different ligands. Hydrodynamic data from diffusion NMR spectroscopy, QELS, and SAXS show that for the aptamer with the full-length terminal stem there is a small amount of structure compaction with DCA binding. For ligand binding by the short terminal stem aptamer, we propose a binding mechanism where secondary structure forms upon DCA binding starting from a free structure where the aptamer exists in a compact form.

Salt-mediated two-site ligand binding by the cocaine-binding aptamer

“…However, if the length of stem one is shortened to three base pairs, such as for MN19 (Figure 1), the aptamer is loosely structured in the absence of target and upon binding the secondary structure rigidifies in a ligand-induced folding process (4,21). This ligand-induced folding mechanism is retained with quinine binding and when the sequence is altered to allow for binding of the steroid deoxycholic acid (DCA) (18,22). The cocaine-binding aptamer has also been shown to function when separated into two strands, referred to as a split-aptamer (3,13,23).…”

Ultra-high frequency piezoelectric aptasensor for the label-free detection of cocaine

“…Perhaps one reason for the lack of aptasensor adoption is the lack of structure conservation in comparison to that of antibodies. Most aptasensors are designed to take advantage of structural differences between the free and complexed state of aptamer receptors Reinstein et al, 2011;Xie and Walton, 2009). However, unlike antibodies whose structure is mostly conserved (at exception of the variable antigen-binding region) aptamer structures highly vary between different aptamers.…”

Characterization of Aptamer-Protein Complexes by X-ray Crystallography and Alternative Approaches

“…Combining these dedicated analytical techniques can provide useful insights into the aptamer target interaction. A recent example for which a combination of these techniques was used (ITC, DLS, NMR and SAXS) is the transformation of a cocaine binding aptamer to one that became specific for deoxycholic acid, by only replacing a single base pair [19]. …”

“…However, obtaining crystal structures of aptamer-target complexes has proven difficult, and only a few co-crystal structures have become available over the years (Table 1). Besides X-ray crystallography, also other techniques, such as nuclear magnetic resonance (NMR), surface plasmon resonance (SPR), analysis using a quartz crystal microbalance (QCM) [17], isothermal titration calorimetry (ITC), Dynamic light scattering (DLS), circular dichroism (CD) [18] and small-angle X-ray scattering (SAXS) [12,19], have been used to study aptamer-target binding. Knowledge on the target-binding mode of newly described aptamers, however, is generally restricted to the affinity of binding.…”