Fast label-free chemiluminescent assay for determination of exonuclease III (ExoIII) activity measured towards hairpin oligonucleotide substrates was developed. The designed substrates consisted of EAD2 aptamer to hemin which was associated with DNA sequence complementary to 5'-terminus fragment of EAD2. In the presence of ExoIII the associated sequence of the hairpin stem was digested, producing EAD2 aptamer which reacted with hemin with the formation of peroxidase-mimicking DNAzyme (PMDNAzyme). The catalytic activity of the produced PMDNAzyme was measured towards luminol/H2O2. Under the optimized conditions the limit of detection and sensitivity of the one-step chemiluminescent assay of ExoIII were 7.3 nM and 1.7 × 108 M-1, respectively. The coefficient of variation (CV) was lower than 6%.

中文翻译：

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用于测定外切核酸酶 III 对发夹寡核苷酸的活性的一步法无标记化学发光试验

开发了用于测定针对发夹寡核苷酸底物测量的外切核酸酶 III (ExoIII) 活性的快速无标记化学发光测定法。设计的底物由与 EAD2 的 5'-末端片段互补的 DNA 序列相关联的血红素的 EAD2 适配体组成。在 ExoIII 存在下，发夹茎的相关序列被消化，产生 EAD2 适体，该适体与血红素反应形成过氧化物酶模拟脱氧核糖核酸酶 (PMDNAzyme)。测量产生的 PMDNAzyme 对鲁米诺/H 2 O 2 的催化活性。在优化条件下，ExoIII 一步化学发光法的检测限和灵敏度分别为 7.3 nM 和 1.7 × 108 M-1。变异系数 (CV) 低于 6%。