Introducing structure-switching functionality into small-molecule-binding aptamers via nuclease-directed truncation

Abstract

We report a broadly applicable enzyme digestion strategy for introducing structure-switching functionality into small-molecule-binding aptamers. This procedure is based on our discovery that exonuclease III (Exo III) digestion of aptamers is greatly inhibited by target binding. As a demonstration, we perform Exo III digestion of a pre-folded three-way-junction (TWJ)-structured cocaine-binding aptamer and a stem–loop-structured ATP-binding aptamer. In the absence of target, Exo III catalyzes 3′-to-5′ digestion of both aptamers to form short, single-stranded products. Upon addition of target, Exo III digestion is halted four bases prior to the target-binding domain, forming a major target-bound aptamer digestion product. We demonstrated that target-binding is crucial for Exo III inhibition. We then determine that the resulting digestion products of both aptamers exhibit a target-induced structure-switching functionality that is absent in the parent aptamer, while still retaining high target-binding affinity. We confirm that these truncated aptamers have this functionality by using an exonuclease I-based digestion assay and further evaluate this characteristic in an electrochemical aptamer-based cocaine sensor and a fluorophore-quencher ATP assay. We believe our Exo III-digestion method should be applicable for the generation of structure-switching aptamers from other TWJ- or stem–loop-containing small-molecule-binding aptamers, greatly simplifying the generation of functionalized sensor elements for folding-based aptasensors.

Engineering base-excised aptamers for highly specific recognition of adenosine

“…To study the binding performance of the A10-excised aptamer, SYBR Green I (SGI) was used for label-free binding assays. [44][45][46][47][48] First, a 50 nM A10-excised aptamer was incubated with 2 mM adenosine ( Fig. 2B).…”

No Structure-Switching Required: A Generalizable Exonuclease-Mediated Aptamer-Based Assay for Small-Molecule Detection

“…27–30 In this work, we found that the binding of small-molecule targets to their respective aptamers can also inhibit exonuclease digestion, and to the best of our knowledge, this is the first work to take advantage of this fact to develop a small-molecule detection assay based on the quantification of aptamer digestion products. We first developed a simple dual-exonuclease-mediated assay that can be generally implemented with unmodified, prefolded aptamers for small-molecule detection.…”

“…We further demonstrated the generality of the dual-exonuclease-mediated assay using an ATP-binding aptamer (ATP-33) 30,40 of which the exact structure of its target-binding domain has not been conclusively determined. 41–43 ATP-33 is prefolded and forms a hairpin structure with a 7-bp blunt-ended stem that is resistant to Exo I digestion (Figure 6A, Exo I).…”

“…27–29 We recently found that this phenomenon also holds true for small-molecule binding aptamers and exploited this finding to introduce structure-switching functionality into fully folded aptamers. 30 Here, we develop an assay that utilizes high-affinity, prefolded aptamers and two exonucleases, Exo III and exonuclease I (Exo I), to detect small molecules. Favorably, this assay does not require any sequence engineering, truncation, or labeling.…”

Label-free profiling of DNA aptamer-small molecule binding using T5 exonuclease

“…To assess whether aptamer–ligand binding inhibits aptamer digestion by T5 Exo, we used a well-characterized 33-nt DNA aptamer that binds to ATP (ATP-33) ( 34 ) derived from the aptamer isolated by Huizenga and Szostak ( 35 ). We digested ATP-33 with T5 Exo in the absence and presence of ATP, and analyzed the digestion process using polyacrylamide gel electrophoresis (PAGE).…”