DNA logic circuits based amplification system for quencher-free and highly sensitive detection of DNA and adenosine triphosphate

Abstract

We fabricated a quencher-free and enzyme-free fluorescence detection system by employing the DNA logic circuits as signal amplifier and 2-aminopurine as signal indicator, and applied it to detect DNA and adenosine triphosphate (ATP). The assay system consisted of three hairpin probes with a sequestered 2-aminopurine molecule in each stem domain which was defined as inputs A, B and C of the logic operation. These three hairpin inputs kept stability and coexisted in reaction solution without target. However, adding target to the system would break the stability and initiate a dynamic assembly of the three inputs through toehold mediated displacement, resulting in the formation of three way junction and the liberation of 2-aminopurine from duplex structure. The structural circumstance changes from duplex to single stand switched the signal from “off” to “on” due to the disarming of base stack interaction, thus attaining amplified fluorescence detection without any extra quencher and avoiding the limitation of distance-independent signal conversion in conventional methods. A limit of detection of 0.46 pM was achieved for target DNA with high discrimination capability. Moreover, the sensing system was expandable for ATP detection. Importantly, the method was simple and easy-to-operate. These features make the DNA logic circuits adaptable as an enzyme-free and quencher-free amplifier, and thus the proposed method offers a powerful platform for DNA and ATP determination, and even other biotargets in clinical diagnosis.