College of Chemistry and Pharmaceutical Sciences, Qingdao Agricultural University , Qingdao 266109, P. R. China.
ACS Appl Mater Interfaces. 2017 Apr 19;9(15):13102-13110. doi: 10.1021/acsami.7b03198. Epub 2017 Apr 10.
Although researches until now have emphasized the influence of an oligonucleotide sequence on the fluorescence of oligonucleotide-stabilized silver nanoclusters (AgNCs), this influence has been explored as a novel ratiometric fluorescent signal transduction in this work. This study builds on our original discovery of a template-transformation phenomenon, which demonstrated that the connection of a special DNA fragment (5'-CACCGCTTT-3') with a green-emitting AgNC nucleation sequence (GNuS, 5'-TGCCTTTTGGGGACGGATA-3') creates a red-emitting AgNC nucleation sequence (RNuS, 5'-CACCGCTTTTGCCTTTTGGGGACGGATA-3'). Attempts to expand this idea and construct elegant ratiometric NanoCluster Beacons (NCBs) for DNA sequence detection are not straightforward, and, thus, we carried out a series of investigations with the goal of understanding the mechanism of this template-transformation phenomenon. Experimental results showed that the six-nucleotide fragment (5'-CACCGC-3') at the 5'-end of RNuS acts as a template convertor and takes full responsibility for the template transformation from GNuS to RNuS. Moreover, we found that the appropriate proximity of the convertor to GNuS also plays a significant role in the template transformation. We then show that the insights gained here for the template-transformation mechanism allow us to construct ratiometric NCBs by simply appending the convertor and the GNuS onto a rationally designed stem-loop probe. This new type of NCB emits intense red fluorescence without the addition of a target DNA and emerges as a new, bright green emission only when hybridized to its target DNA. By measuring the distinct variation in the fluorescence intensity ratios of green and red emission, this ratiometric NCB was demonstrated to sensitively detect Hepatitis-A virus gene sequences, a proof-of-concept target in this work, with good selectivity.
虽然迄今为止的研究强调了寡核苷酸序列对寡核苷酸稳定的银纳米簇（AgNCs）荧光的影响，但在这项工作中，这种影响被探索为一种新的比率荧光信号转导。本研究基于我们最初发现的模板转化现象，该现象表明，将特殊 DNA 片段（5'-CACCGCTTT-3'）与绿色发射 AgNC 成核序列（GNuS，5'-TGCCTTTTGGGGACGGATA-3'）连接，会产生红色发射 AgNC 成核序列（RNuS，5'-CACCGCTTTTGCCTTTTGGGGACGGATA-3'）。尝试扩展这个想法并构建用于 DNA 序列检测的优雅比率纳米簇信标（NCB）并不简单，因此，我们进行了一系列研究，旨在理解这种模板转化现象的机制。实验结果表明，RNuS 5'-端的六核苷酸片段（5'-CACCGC-3'）作为模板转化器，对 GNuS 到 RNuS 的模板转化负全部责任。此外，我们发现转化器与 GNuS 的适当接近度也对模板转化起着重要作用。然后，我们表明，这里获得的关于模板转化机制的见解使我们能够通过简单地将转化器和 GNuS 添加到合理设计的茎环探针上来构建比率 NCB。这种新型 NCB 在没有添加靶 DNA 的情况下会发出强烈的红色荧光，只有与靶 DNA 杂交时才会出现新的亮绿色发射。通过测量绿色和红色发射荧光强度比的明显变化，该比率 NCB 被证明能够灵敏地检测肝炎病毒基因序列，这是本工作中的一个概念验证目标，具有良好的选择性。
