Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources, Ministry of Education of China, College of Chemistry and Chemical Engineering, Guangxi Normal University, Guilin, 541004, China.
Anal Chem. 2011 Dec 1;83(23):8913-8. doi: 10.1021/ac2013114. Epub 2011 Nov 10.
DNA cleavage by endonucleases plays an important role in many biological events such as DNA replication, recombination, and repair and is used as a powerful tool in medicinal chemistry. However, conventional methods for assaying endonuclease activity and inhibition by gel electrophoresis and chromatography techniques are time-consuming, laborious, not sensitive, or costly. Herein, we combine the high specificity of DNA cleavage reactions with the benefits of quantum dots (QDs) and ultrahigh quenching abilities of inter- and intramolecular quenchers to develop highly sensitive and specific nanoprobes for multiplexed detection of endonucleases. The nanoprobe was prepared by conjugating two sets of DNA substrates carrying quenchers onto the surface of aminated QDs through direct assembly and DNA hybridization. With this new design, the background fluorescence was significantly suppressed by introducing inter- and intramolecular quenchers. When these nanoprobes are exposed to the targeted endonucleases, specific DNA cleavages occur and pieces of DNA fragments are released from the QD surface along with the quenchers, resulting in fluorescence recovery. The endonuclease activity was quantified by monitoring the change in the fluorescence intensity. The detection was accomplished with a single excitation light. Multiplexed detection was demonstrated by simultaneously assaying EcoRI and BamHI (as model analytes) using two different emissions of QDs. The limits of detection were 4.0 × 10(-4) U/mL for EcoRI and 8.0 × 10(-4) U/mL for BamHI, which were at least 100 times more sensitive than traditional gel electrophoresis and chromatography assays. Moreover, the potential application of the proposed method for screening endonuclease inhibitors has also been demonstrated. The assay protocol presented here proved to be simple, sensitive, effective, and easy to carry out.
核酸内切酶的 DNA 切割在许多生物事件中发挥着重要作用，例如 DNA 复制、重组和修复，并且被用作医学化学中的强大工具。然而，用于测定内切酶活性和抑制作用的常规方法，如凝胶电泳和色谱技术，耗时、费力、不灵敏或昂贵。在此，我们将 DNA 切割反应的高特异性与量子点 (QDs) 的优势以及分子内和分子间猝灭剂的超高猝灭能力相结合，开发了用于多重检测内切酶的高灵敏度和特异性纳米探针。该纳米探针通过直接组装和 DNA 杂交将两组带有猝灭剂的 DNA 底物连接到氨基化 QD 的表面来制备。通过这种新设计，通过引入分子内和分子间猝灭剂，显著抑制了背景荧光。当这些纳米探针暴露于靶向内切酶时，会发生特定的 DNA 切割，并且 DNA 片段与猝灭剂一起从 QD 表面释放出来，从而导致荧光恢复。通过监测荧光强度的变化来定量测定内切酶的活性。通过使用单个激发光来完成检测。通过同时使用两种不同的 QD 发射来同时检测 EcoRI 和 BamHI（作为模型分析物）来实现多重检测。EcoRI 的检测限为 4.0×10(-4)U/mL，BamHI 的检测限为 8.0×10(-4)U/mL，比传统的凝胶电泳和色谱分析方法至少灵敏 100 倍。此外，还证明了该方法在筛选内切酶抑制剂方面的潜在应用。所提出的方法证明简单、灵敏、有效且易于实施。
