Colorimetric Sensing by Using Allosteric-DNAzyme-Coupled Rolling Circle Amplification and a Peptide Nucleic Acid-Organic Dye Probe
Colorimetric Sensing by Using Allosteric-DNAzyme-Coupled Rolling Circle Amplification and a Peptide Nucleic Acid-Organic Dye Probe
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DOI：
10.1002/anie.200805966
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发表时间：
2009-01-01
影响因子：
16.6
通讯作者：
Li, Yingfu
中科院分区：
文献类型：
--
作者：
Ali, M. Monsur;Li, Yingfu
DNA适体和DNA酶是近年来化学生物学研究的热点。[1]这两类合成DNA分子可以通过体外选择从随机序列DNA库中分离出来，[2]被认为是抗体和酶的有吸引力的替代品。特别的优点是DNA的更大的化学稳定性和其通过自动化合成的简单制备。已经产生了大量的DNA适体用于识别从小分子（如三磷酸腺苷（ATP））到蛋白质（如凝血酶）和复杂分子组装体（如细胞）的靶标。[3]同样，许多DNA酶已经被合成用于催化不同的化学反应，例如DNA和RNA的切割和连接。[4]最近，变构核酶[5]的概念已经适用于变构DNA酶的设计，其中DNA适体以这样的方式连接到DNA酶，使得DNA酶只能被与适体结合的配体激活。[6]变构DNA酶作为生物传感工具是令人感兴趣的，因为适体与其特异性配体之间的分子识别事件可以被翻译成用于信号产生和放大的DNA酶的活性。滚环扩增（RCA）是一种简单的酶促过程，可用于产生具有串联重复序列的非常长的单链DNA（ssDNA）分子。[7]这个过程是用一个短的DNA引物和一个环状模板，在等温条件下，通过特殊的DNA聚合酶，如f29 DNA聚合酶，具有链置换能力。RCA传统上用于DNA的灵敏检测。[8]然而，近年来，RCA已经通过使用DNA适体和变构DNA酶扩展到检测其他靶标，例如蛋白质和小分子。[9]例如，Willner和Mao的研究小组最近使用RCA技术来产生报告DNA酶的重复单元，用于DNA的高灵敏度检测。[9e Ellington及其同事创建了一种配体依赖性连接酶DNAzyme，可以生成环状DNA模板以启动RCA过程，作为检测小分子靶标[9d]和蛋白质的一种方式。[9b]然而，RCA和功能性DNA用于以比色形式感测非核酸靶标的用途尚未得到证实，并且是当前研究的焦点。我们相信这样的测定将显著地扩展功能性核酸的实际效用。我们的策略如图1所示。实现了三个关键设计：RNA切割变构DNA酶，RCA，和基于肽核酸（PNA）和有机染料的比色报告机制。在存在预期靶标的情况下，变构DNA酶切割特殊的含RNA底物并释放DNA分子，该DNA分子可被f29 DNA聚合酶用作引物以启动RCA反应以产生长ssDNA分子。然后在DiSC 2（5）（3，3 '-二乙基硫杂双环菁）存在下与互补PNA杂交后，用比色法检测RCA产物。已知PNA分子与互补DNA序列形成高度稳定的双链体结构。[10]还报道了DiSC 2（5）在与DNA/PNA双链体结合时从蓝色变为紫色。这
DNA aptamers and DNAzymes have recently received considerable attention in chemical-biology research.[1] These two classes of synthetic DNA molecules, which can be isolated from random-sequence DNA pools by in vitro selection,[2] are regarded as attractive alternatives to antibodies and enzymes. Particular advantages are the greater chemical stability of DNA and its straightforward preparation by automated synthesis. A large number of DNA aptamers have been produced for the recognition of targets ranging from small molecules (such as adenosine triphosphate (ATP)) to proteins (such as thrombin) and complex molecular assemblies (such as cells).[3] Likewise, many DNAzymes have been synthesized for the catalysis of diverse chemical reactions, such as the cleavage and ligation of DNA and RNA.[4] More recently, the concept of allosteric ribozymes [5] has been adapted to the design of allosteric DNAzymes, in which a DNA aptamer is connected to a DNAzyme in such a way that the DNAzyme can only be activated by the ligand that binds to the aptamer.[6] Allosteric DNAzymes are interesting as biosensing tools, because the molecular-recognition event between an aptamer and its specific ligand can be translated into the activity of a DNAzyme for signal generation and amplification. Rolling circle amplification (RCA) is a simple enzymatic process that can be used to generate very long single-stranded DNA (ssDNA) molecules with tandem repeats.[7] This process is carried out with a short DNA primer and a circular template under isothermal conditions by special DNA polymerases, such as f29 DNA polymerase, with stranddisplacement abilities. RCA has traditionally been used for the sensitive detection of DNA.[8] In recent years, however, RCA has been extended to the detection of other targets, such as proteins and small molecules, through the use of DNA aptamers and allosteric DNAzymes.[9] For example, the research groups of Willner and Mao recently used the RCA technique to generate repetitive units of a reporter DNAzyme for the highly sensitive detection of DNA.[9e, f] Ellington and co-workers created a ligand-dependent ligase DNAzyme that can generate a circular DNA template to initiate an RCA process as a way to detect small-molecule targets [9d] and proteins.[9b] However, the use of RCA and functional DNA for the sensing of non-nucleic-acid targets in a colorimetric format has yet to be demonstrated and was the focus of the current study. We believe such assays will expand the practical utility of functional nucleic acids significantly. Our strategy is illustrated in Figure 1. Three key designs are implemented: an RNA-cleaving allosteric DNAzyme, RCA, and a colorimetric reporting mechanism based on a peptide nucleic acid (PNA) and an organic dye. In the presence of the intended target, the allosteric DNAzyme cleaves a special RNA-containing substrate and releases a DNA molecule that can be used by f29 DNA polymerase as the primer to initiate an RCA reaction for the generation of a long ssDNA molecule. The RCA products are then detected colorimetrically upon hybridization with a complementary PNA in the presence of DiSC2 (5)(3, 3’-diethylthiadicarbocyanine). PNA molecules are known to form highly stable duplex structures with complementary DNA sequences.[10] It has also been reported that DiSC2 (5) changes color from blue to purple upon binding to a DNA/PNA duplex. This