Aptamer Switch Probe Based on Intramolecular Displacement

TLDR

The authors introduce the “aptamer switch probe” (ASP), a novel aptamer‑based molecular probe. The ASP consists of an aptamer linked via a PEG spacer to a short complementary DNA strand, with a fluorophore and quencher at opposite ends; in the absence of target the DNA hybridizes to the aptamer to quench fluorescence, while target binding disrupts the hybridization and restores fluorescence. ATP and thrombin ASPs showed quantitative fluorescence enhancement with target concentration, high selectivity, rapid response, and promising suitability for reusable, stable, sensitive biochip and in‑situ imaging applications.

Abstract

A novel aptamer-based molecular probe design employing intramolecular signal transduction is demonstrated. The probe is composed of three elements: an aptamer, a short, partially cDNA sequence, and a PEG linker conjugating the aptamer with the short DNA strand. We have termed this aptamer probe an "aptamer switch probe", or ASP. The ASP design utilizes both a fluorophore and a quencher which are respectively modified at the two termini of the ASP. In the absence of the target molecule, the short DNA will hybridize with the aptamer, keeping the fluorophore and quencher in close proximity, thus switching off the fluorescence. However, when the ASP meets its target, the binding between the aptamer and the target molecule will disturb the intramolecular DNA hybridization, move the quencher away from the fluorophore, and, in effect, switch on the fluorescence. Both ATP and human α-thrombin aptamers were engineered to demonstrate this design, and both showed that fluorescence enhancement could be quantitatively mediated by the addition of various amounts of target molecules. Both of these ASPs presented excellent selectivity and prompt response toward their targets. With intrinsic advantages resulting from its intramolecular signal transduction architecture, the ASP design holds promising potential for future applications, such as biochip and in situ imaging, which require reusability, excellent stability, prompt response, and high sensitivity.