Ultrasensitive detection of 3′-5′ exonuclease enzymatic activity using molecular beacons

Abstract

An ultrasensitive and rapid turn-on fluorescence assay has been developed for the detection of 3′-5′ exonuclease activity of exonuclease III (Exo III) using molecular beacons (MBs). This method has a linear detection range from 0.04 to 8.00 U mL⁻¹ with a limit of detection of 0.01 U mL⁻¹. In order to improve the selectivity of the method, a dual-MB system has been developed to distinguish between different exonucleases. With the introduction of two differently designed MBs which respond to different exonucleases, the T5 exonuclease, Exo III and RecJf exonucleases can be easily distinguished from each other. Furthermore, fetal bovine serum and fresh mouse serum were used as complex samples to investigate the feasibility of the dual-MB system for the detection of the enzymatic activity of Exo III. As a result, the dual-MB system showed a similar calibration curve for the detection of Exo III as in the ideal buffer solution. The designed MB probe could be a potential sensor for the detection of Exo III in biological samples.