A ligation-triggered DNAzyme cascade for amplified fluorescence detection of biological small molecules with zero-background signal.
A ligation-triggered DNAzyme cascade for amplified fluorescence detection of biological small molecules with zero-background signal.
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DOI：
10.1021/ja203693b
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发表时间：
2011-08-03
影响因子：
15
通讯作者：
Tan W
中科院分区：
文献类型：
--
作者：
Lu LM;Zhang XB;Kong RM;Yang B;Tan W
许多类型的荧光传感系统已被报道用于生物小分子。特别是，已经开发了几种方法用于识别ATP或NAD+，但它们仅显示出中等的灵敏度，并且它们不能将ATP或NAD+与它们各自的类似物区分开。我们已经解决了这些限制，并在这里报告的双重战略，结合分裂DNA酶为基础的背景减少与催化和分子信标（CAMB）为基础的扩增检测开发连接触发的DNA酶级联反应，从而导致DNA酶的敏感性。首先，将8-17 DNA酶分成两个单独的寡核苷酸片段作为DNA连接反应的结构单元，从而提供零背景信号以提高总体灵敏度。接下来，CAMB策略进一步用于通过循环和再生DNA酶实现的扩增信号检测，以实现催化信标的真正酶促多重转换（一种酶催化几种底物的切割）。这种零背景信号和信号放大的组合显著提高了传感系统的灵敏度，导致ATP和NAD+的检测限分别为100和50 pM，远低于先前报道的生物传感器。此外，通过利用DNA连接反应的高度特异性生物分子依赖性，开发的DNAzyme级联显示出对靶辅因子（ATP或NAD+）的显著高选择性，并且靶生物小分子可以与其类似物区分开。因此，作为一种新的和通用的平台，设计的DNA连接反应为基础的传感系统，这种新的连接触发DNAzyme级联方法可能会发现在环境和生物医学领域的广泛应用。
Many types of fluorescent sensing systems have been reported for biological small molecules. Particularly, several methods have been developed for the recognition of ATP or NAD+, but they only show moderate sensitivity, and they cannot discriminate either ATP or NAD+ from their respective analogues. We have addressed these limitations and report here a dual strategy which combines split DNAzyme-based background reduction with catalytic and molecular beacon (CAMB)-based amplified detection to develop a ligation-triggered DNAzyme cascade, resulting in ultrahigh sensitivity. First, the 8–17 DNAzyme is split into two separate oligonucleotide fragments as the building blocks for the DNA ligation reaction, thereby providing a zero-background signal to improve overall sensitivity. Next, a CAMB strategy is further employed for amplified signal detection achieved through cycling and regenerating the DNAzyme to realize the true enzymatic multiple turnover (one enzyme catalyzes the cleavage of several substrates) of catalytic beacons. This combination of zero-background signal and signal amplification significantly improves the sensitivity of the sensing systems, resulting in detection limits of 100 and 50 pM for ATP and NAD+, respectively, much lower than those of previously reported biosensors. Moreover, by taking advantage of the highly specific biomolecule-dependence of the DNA ligation reaction, the developed DNAzyme cascades show significantly high selectivity toward the target cofactor (ATP or NAD+), and the target biological small molecule can be distinguished from its analogues. Therefore, as a new and universal platform for the design of DNA ligation reaction-based sensing systems, this novel ligation-triggered DNAzyme cascade method may find a broad spectrum of applications in both environmental and biomedical fields.
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