Nano Medical Engineering Laboratory, RIKEN Advanced Science Institute , 2-1, Hirosawa, Wako-Shi, Saitama 351-0198, Japan.
J Am Chem Soc. 2013 Sep 25;135(38):14172-8. doi: 10.1021/ja404743m. Epub 2013 Sep 9.
Oligonucleotide-templated reactions are powerful tools for the detection of nucleic acid sequences. One of the major scientific challenges associated with this technique is the rational design of non-enzyme-mediated catalytic templated reactions capable of multiple turnovers that provide high levels of signal amplification. Herein, we report the development of a nucleophilic aromatic substitution reaction-triggered fluorescent probe. The probe underwent a rapid templated reaction without any of the undesired background reactions. The fluorogenic reaction conducted in the presence of a template provided a 223-fold increase in fluorescence after 30 s compared with the nontemplated reaction. The probe provided an efficient level of signal amplification that ultimately enabled particularly sensitive levels of detection. Assuming a simple model for the templated reactions, it was possible to estimate the rate constants of the chemical reaction in the presence and in the absence of the template. From these kinetic analyses, it was possible to confirm that an efficient turnover cycle had been achieved, on the basis of the dramatic enhancement in the rate of the chemical reaction considered to be the rate-determining step. With maximized turnover efficiency, it was demonstrated that the probe could offer a high turnover number of 1500 times to enable sensitive levels of detection with a detection limit of 0.5 pM in the catalytic templated reactions.
寡核苷酸模板反应是检测核酸序列的有力工具。与该技术相关的主要科学挑战之一是合理设计能够进行多次周转的非酶介导催化模板反应，从而提供高水平的信号放大。在此，我们报告了一种亲核芳香取代反应触发的荧光探针的开发。该探针在模板存在下发生快速模板反应，而没有任何不需要的背景反应。在模板存在下进行的荧光反应在 30 秒后与非模板反应相比，荧光增加了 223 倍。该探针提供了高效的信号放大水平，最终实现了特别灵敏的检测水平。假设模板反应的简单模型，可以估计在有和没有模板的情况下化学反应的速率常数。通过这些动力学分析，可以确认已经实现了有效的周转循环，这是基于被认为是速率决定步骤的化学反应速率的急剧增强。通过最大化的周转效率，证明探针可以提供高达 1500 次的高周转数，从而能够在催化模板反应中实现灵敏的检测水平，检测限为 0.5 pM。
