This study presents a technique for detecting 3′–5′ exonuclease activity through the use of CRISPR/Cas12a. These enzymes, including 3′–5′ exonuclease (Exo III), perform crucial roles in various cellular processes and are associated with life expectancy. However, imbalances in their expression can increase susceptibility to diseases such as cancer, particularly under prolonged stress. In this study, an activator sequence of CRISPR/Cas12a was constructed on the 5′–end of a hairpin probe (HP), forming a blunt end. When the 3′–end of the HP was hydrolyzed with Exo III activity, the activator sequence of Cas12a was exposed, which led to collateral cleavage of the DNA signal probe and generated a fluorescent signal, allowing sensitive and highly specific Exo III detection. This detection principle relied on the fact that Exo III exclusively cleaves the 3′–end mononucleotide of dsDNA and does not affect ssDNA. Based on this strategy, Exo III activity was successfully assayed at 0.0073 U/mL, demonstrating high sensitivity. In addition, this technique was used to screen candidate inhibitors of Exo III activity.

We present a novel label-free colorimetric method for detecting exonuclease III (Exo III) activity using the peroxidase-mimicking activity of cerium oxide nanoparticles (nanoceria). Exo III, an enzyme that specifically catalyzes the stepwise removal of mononucleotides from the 3′-OH termini of double-stranded DNA, plays a significant role in various cellular and physiological processes, including DNA proofreading and repair. Malfunctions of Exo III have been associated with increased cancer risks. To assay the activity of Exo III, we applied the previous reports in that the peroxidase-mimicking activity of nanoceria is inhibited due to the aggregation induced by the electrostatic attraction between DNA and nanoceria. In the presence of Exo III, the substrate DNA (subDNA), which inhibits nanoceria’s activity, is degraded, thereby restoring the peroxidase-mimicking activity of nanoceria. Consequently, the 3,3′,5,5′-tetramethylbenzidine (TMB) substrate is oxidized, leading to a color change from colorless to blue, along with an increase in the absorbance intensity. This approach enabled us to reliably detect Exo III at a limit of detection (LOD) of 0.263 units/mL across a broad dynamic range from 3.1 to 400 units/mL, respectively, with an outstanding specificity. Since this approach does not require radiolabels, complex DNA design, or sophisticated experimental techniques, it provides a simpler and more feasible alternative to standard methods.