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首页> 外文期刊>Chemistry: A European journal >Nucleic Acid Templated Reactions: Consequences of Probe Reactivity and Readout Strategy for Amplified Signaling and Sequence Selectivity
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Nucleic Acid Templated Reactions: Consequences of Probe Reactivity and Readout Strategy for Amplified Signaling and Sequence Selectivity

机译：核酸模板化反应：探针反应性和扩增信号和序列选择性读出策略的后果

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摘要

DNA- and RNA-templated chemical reactions can serve as a diagnostic means for the detection of nucleic acids. Reaction schemes that allow amplified detection are of high interest for polymerase chain reaction (PCR)free DNA and RNA diagnosis. These reactions typically draw upon the catalytic activity of the template, which is able to trigger the conversion of many signaling molecules per template molecule. However, the design of reactive probes that allow both sensitive and selective nucleic acid detection is a challenge and requires insight into three major parameters: a) reactivity Of functional groups involved, b) affinity of probes for the template, and c) the readout system. In this study We used peptide nucleic acid (PNA)-based probes to investigate in detail the signaling power and the selectivity of a transfer reaction derived from a native chemical ligation. We show that subtle variations of the thioesters involved had a tremendous impact on the sensitivity and selectivity of: the reaction system. The results suggest that reactions at turnover conditions require low rates of non-templated reaction pathways to provide high target selectivity and sensitivity. Oil the other hand, very high rates of templated reactions should be avoided to allow mismatched probe-template complexes to dissociate prior to bond formation. Furthermore, the temperature dependence of the DNA-catalyzed transfer reaction was studied and provided insight into crucial strand-exchange processes. Further improvements of selective signaling were achieved through a new readout based on pyrene-transfer reactions. This method reduces background signals and enables significant increases in the signaling rates compared with previous fluorescence-based methods.
机译：以DNA和RNA为模板的化学反应可以作为检测核酸的诊断手段。允许进行扩增检测的反应方案对于无聚合酶链反应（PCR）的DNA和RNA诊断非常重要。这些反应通常利用模板的催化活性，该催化活性能够触发每个模板分子许多信号分子的转化。但是，允许同时进行灵敏和选择性核酸检测的反应性探针的设计是一项挑战，需要深入了解三个主要参数：a）涉及的官能团的反应性，b）探针对模板的亲和力，以及c）读出系统。在这项研究中，我们使用了基于肽核酸（PNA）的探针来详细研究源自天然化学连接的信号传递能力和转移反应的选择性。我们表明所涉及的硫酯的细微变化对反应体系的灵敏度和选择性产生了巨大影响。结果表明，在周转条件下的反应需要低比率的非模板反应路径来提供高目标选择性和灵敏度。另一方面，应避免非常高的模板化反应速率，以使错配的探针-模板复合物在形成键之前解离。此外，对DNA催化的转移反应的温度依赖性进行了研究，并为关键的链交换过程提供了见识。通过基于pyr转移反应的新读数实现了选择性信号转导的进一步改进。与以前的基于荧光的方法相比，该方法可减少背景信号并显着提高信号传输速率。

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