Sensitive and Selective Amplified Fluorescence DNA Detection Based on Exonuclease III-Aided Target Recycling

TLDR

Traditional oligonucleotide detection methods such as molecular beacons suffer from a 1:1 target–probe hybridization ratio, limiting their sensitivity. We present a nuclease‑amplified detection scheme that uses exonuclease III to recycle target strands, enabling high sensitivity without restricting target choice. The assay detects target DNA down to 10 pM at 37 °C, 20 aM at 4 °C (with a 24‑h incubation), and can be read visually, making it suitable for low‑resource settings.

Abstract

A limitation of many traditional approaches to the detection of specific oligonucleotide sequences, such as molecular beacons, is that each target strand hybridizes with (and thus activates) only a single copy of the relevant probe sequence. This 1:1 hybridization ratio limits the gain of most approaches and thus their sensitivity. Here we demonstrate a nuclease-amplified DNA detection scheme in which exonuclease III is used to "recycle" target molecules, thus leading to greatly improved sensitivity relative to, for example, traditional molecular beacons without any significant restriction in the choice of target sequences. The exonuclease-amplified assay can detect target DNA at concentrations as low as 10 pM when performed at 37 °C, which represents a significant improvement over the equivalent molecular beacon alone. Moreover, at 4 °C we can obtain a detection limit as low as 20 aM, albeit at the cost of a 24 h incubation period. Finally, our assay can be easily interrogated with the naked eye and is thus amenable to deployment in the developing world, where fluorometric detection is more problematic.