Material & Component Convergence R&D Department, Korea Institute of Industrial Technology (KITECH), Ansan 15588, Republic of Korea.
Department of Chemistry, Gangneung-Wonju National University, Gangneung 25457, Republic of Korea.
Int J Mol Sci. 2023 Aug 2;24(15):12330. doi: 10.3390/ijms241512330.
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.
我们提出了一种新颖的无标记比色法，用于检测外切核酸酶 III（Exo III）的活性，该方法利用了氧化铈纳米粒子（nanoceria）的过氧化物酶模拟活性。Exo III 是一种特异性地催化从双链 DNA 的 3' -OH 末端逐步去除单核苷酸的酶，在各种细胞和生理过程中发挥着重要作用，包括 DNA 校对和修复。Exo III 的功能障碍与癌症风险增加有关。为了测定 Exo III 的活性，我们应用了之前的报道，即由于 DNA 和氧化铈纳米粒子之间的静电吸引引起的聚集，氧化铈纳米粒子的过氧化物酶模拟活性被抑制。在 Exo III 的存在下，抑制氧化铈纳米粒子活性的底物 DNA（subDNA）被降解，从而恢复氧化铈纳米粒子的过氧化物酶模拟活性。因此，3,3',5,5'-四甲基联苯胺（TMB）底物被氧化，导致颜色从无色变为蓝色，同时吸光度强度增加。这种方法能够可靠地检测到 Exo III，其检测限（LOD）为 0.263 单位/mL，在 3.1 至 400 单位/mL 的宽动态范围内，具有出色的特异性。由于这种方法不需要放射性标记物、复杂的 DNA 设计或复杂的实验技术，因此为标准方法提供了一种更简单、更可行的替代方法。
