Zavoiura Oleksandr, Resch-Genger Ute, Seitz Oliver
Division Biophotonics, Federal Institute for Materials Research and Testing (BAM), Berlin, Germany.
Department of Chemistry, Humboldt University of Berlin, Berlin, Germany.
Methods Mol Biol. 2020;2105:187-198. doi: 10.1007/978-1-0716-0243-0_11.
Oligonucleotide-templated reactions (OTRs) between two reactive hybridization probes allow for the detection of a DNA or RNA of interest by exploiting the target molecule as a catalyst of chemical reactions. The product of such a reaction commonly exhibits distinct fluorescence properties and can be detected by the means of fluorescence spectroscopy. The vast majority of OTR systems utilize organic dyes as fluorescent reporters. However, the use of brighter emitters, such as semiconductor quantum dots (QDs), has potential to improve the sensitivity of detection by providing brighter signals and permitting the use of probes at very low concentrations. Here we report an RNA-templated reaction between two fluorescently labeled peptide nucleic acid (PNA)-based probes, which proceeds on the surface of a QD. The QD-bound PNA probe bears a cysteine functionality, while the other PNA is functionalized with an organic dye as a thioester. OTR between these probes proceeds through a transfer of the organic dye to the QD and can be conveniently monitored via fluorescence resonance energy transfer (FRET) from the QD to the Cy5. The reaction was performed in a conventional fluorescence microplate reader and permits the detection of RNA in the picomolar range.
两个反应性杂交探针之间的寡核苷酸模板反应（OTR），通过利用目标分子作为化学反应的催化剂，可实现对感兴趣的DNA或RNA的检测。此类反应的产物通常具有独特的荧光特性，可通过荧光光谱法进行检测。绝大多数OTR系统使用有机染料作为荧光报告基团。然而，使用更亮的发光体，如半导体量子点（QD），有可能通过提供更亮的信号并允许在极低浓度下使用探针来提高检测灵敏度。在此，我们报道了两种基于荧光标记肽核酸（PNA）的探针之间的RNA模板反应，该反应在量子点表面进行。与量子点结合的PNA探针带有半胱氨酸官能团，而另一种PNA则用有机染料作为硫酯进行功能化。这些探针之间的OTR通过有机染料向量子点的转移而进行，并且可以通过从量子点到Cy5的荧光共振能量转移（FRET）方便地进行监测。该反应在传统的荧光酶标仪中进行，可实现皮摩尔范围内RNA的检测。
