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Simple and convenient G-quadruplex-based fluorescence assay of micrococcal nuclease activity

机译：简单，方便的基于G-四链体的微球菌核酸酶活性的荧光测定

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摘要

As the extracellular nuclease of Staphylococcus aureus (S. aureus), micrococcal nuclease (MNase), which preferentially digests single-stranded nucleic acids, can be used as the standard to identify S. aureus and can be used to evaluate the pathogenicity of S. aureus. So the assay of MNase is of high importance. However, traditional methods for the assay of MNase activity have intrinsic limitations such as sophisticated synthesis processes, the need for functionalizing thiol (or dye)-modified oligonucleotide probes or critical operation conditions. Herein, a simple and convenient fluorescence sensing platform for MNase activity has been developed based on N-methyl mesoporphyrin IX (NMM)/G-quadruplexes. In the absence of MNase, the G-rich single stranded DNA (ssDNA) folds into a quadruplex in the presence of monovalent ions, thus greatly enhancing the fluorescence of NMM (a specific G-quadruplex binder). In the presence of MNase, the G-rich ssDNA was digested into small fragments. As a result, the fluorescence of the solution decreases with increase of MNase activity. Under the optimized conditions, the fluorescence intensity exhibits a linear correlation to MNase concentration in a wide range of 1.2 x 10(-4)-2.4 x 10(-3) units per mL with a detection limit of 7.1 x 10(-5) units per mL and good selectivity. The detection limit is much better or at least comparable to previous reports. Given its simplicity, easy operation, sensitivity and cost-effectiveness, this method can be extended to other nuclease assays.
机译：作为金黄色葡萄球菌（S. aureus）的细胞外核酸酶，优先消化单链核酸的微球菌核酸酶（MNase）可作为鉴定金黄色葡萄球菌的标准品，并可用于评估S.的致病性。金黄色的。因此，MNase的检测非常重要。但是，用于测定MNase活性的传统方法具有固有的局限性，例如复杂的合成过程，功能化巯基（或染料）修饰的寡核苷酸探针的功能或关键操作条件。在此，基于N-甲基中卟啉IX（NMM）/ G-四链体，开发了一种用于MNase活性的简单方便的荧光传感平台。在缺少MNase的情况下，富含G的单链DNA（ssDNA）在存在单价离子的情况下会折叠成四链体，从而大大增强了NMM（一种特定的G-四链体结合剂）的荧光。在MNase存在下，富含G的ssDNA被消化成小片段。结果，溶液的荧光随着MNase活性的增加而降低。在最佳条件下，荧光强度与MNase浓度呈线性相关，范围为每毫升1.2 x 10（-4）-2.4 x 10（-3）单位/ mL，检出限为7.1 x 10（-5）单位/ mL和良好的选择性。检出限要好得多，至少可以与以前的报告相比。鉴于其简单，易操作，灵敏性和成本效益，该方法可以扩展到其他核酸酶检测。

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