State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, PR China.
Chemistry. 2012 Mar 26;18(13):3992-9. doi: 10.1002/chem.201102290. Epub 2012 Feb 15.
The DNA nick repair catalyzed by DNA ligase is significant for fundamental life processes, such as the replication, repair, and recombination of nucleic acids. Here, we have employed ligase to regulate DNAzyme activity and developed a homogeneous, colorimetric, label-free and DNAzyme-based strategy to detect DNA ligase activity. This novel strategy relies on the ligation-trigged activation or production of horseradish peroxidase mimicking DNAzyme that catalyzes the generation of a color change signal; this results in a colorimetric assay of DNA ligase activity. Using T4 DNA ligase as a model, we have proposed two approaches to demonstrate the validity of the DNAzyme strategy. The first approach utilizes an allosteric hairpin-DNAzyme probe specifically responsive to DNA ligation; this approach has a wide detection range from 0.2 to 40 U mL(-1) and a detection limit of 0.2 U mL(-1). Furthermore, the approach was adapted to probe nucleic acid phosphorylation and single nucleotide mismatch. The second approach employs a "split DNA machine" to produce numerous DNAzymes after being reassembled by DNA ligase; this greatly enhances the detection sensitivity by a signal amplification cascade to achieve a detection limit of 0.01 U mL(-1).
DNA 连接酶催化的 DNA 缺口修复对于核酸的复制、修复和重组等基本生命过程至关重要。在这里，我们利用连接酶来调节 DNA 酶的活性，并开发了一种均相、比色、无标记且基于 DNA 酶的策略来检测 DNA 连接酶活性。这种新策略依赖于连接触发的 DNA 酶的激活或产生，该酶可催化产生颜色变化信号；这导致了 DNA 连接酶活性的比色测定。我们使用 T4 DNA 连接酶作为模型，提出了两种方法来证明 DNA 酶策略的有效性。第一种方法利用一种变构发夹 DNA 酶探针，该探针特异性响应 DNA 连接；这种方法的检测范围很宽，从 0.2 到 40 U/ml(-1)，检测限为 0.2 U/ml(-1)。此外，该方法被用于探测核酸磷酸化和单核苷酸错配。第二种方法采用“分裂 DNA 机器”，在 DNA 连接酶重新组装后产生大量 DNA 酶；通过信号放大级联反应，大大提高了检测灵敏度，检测限达到 0.01 U/ml(-1)。
