Barnoin Guillaume, Shaya Janah, Richert Ludovic, Le Hoang-Ngoan, Vincent Steve, Guérineau Vincent, Mély Yves, Michel Benoît Y, Burger Alain
Université Côte d'Azur, CNRS, Institut de Chimie de Nice, UMR 7272 - Parc Valrose, 06108 Nice cedex 2, France.
Laboratoire de Biophotonique et Pharmacologie, UMR 7213 CNRS, Université de Strasbourg, Faculté de pharmacie, 74 Route du Rhin, 67401 Illkirch, France.
Nucleic Acids Res. 2021 Jul 9;49(12):e72. doi: 10.1093/nar/gkab237.
The sensitivity of FRET-based sensing is usually limited by the spectral overlaps of the FRET donor and acceptor, which generate a poor signal-to-noise ratio. To overcome this limitation, a quenched donor presenting a large Stokes shift can be combined with a bright acceptor to perform Dark Resonance Energy Transfer (DRET). The consequent fluorogenic response from the acceptor considerably improves the signal-to-noise ratio. To date, DRET has mainly relied on a donor that is covalently bound to the acceptor. In this context, our aim was to develop the first intermolecular DRET pair for specific sensing of nucleic acid sequences. To this end, we designed DFK, a push-pull probe based on a fluorenyl π-platform that is strongly quenched in water. DFK was incorporated into a series of oligonucleotides and used as a DRET donor with Cy5-labeled complementary sequences. In line with our expectations, excitation of the dark donor in the double-labeled duplex switched on the far-red Cy5 emission and remained free of cross-excitation. The DRET mechanism was supported by time-resolved fluorescence measurements. This concept was then applied with binary probes, which confirmed the distance dependence of DRET as well as its potency in detecting sequences of interest with low background noise.
基于荧光共振能量转移（FRET）的传感灵敏度通常受FRET供体与受体光谱重叠的限制，这会导致信噪比不佳。为克服这一限制，可将具有大斯托克斯位移的猝灭供体与明亮的受体结合，以进行暗共振能量转移（DRET）。受体随之产生的荧光响应可显著提高信噪比。迄今为止，DRET主要依赖与受体共价结合的供体。在此背景下，我们的目标是开发首个用于特异性检测核酸序列的分子间DRET对。为此，我们设计了DFK，这是一种基于芴基π平台的推挽式探针，在水中强烈猝灭。DFK被整合到一系列寡核苷酸中，并用作与Cy5标记的互补序列的DRET供体。与我们的预期一致，在双标记双链体中对暗供体的激发开启了远红Cy5发射，且不存在交叉激发。时间分辨荧光测量结果支持了DRET机制。然后将这一概念应用于二元探针，证实了DRET的距离依赖性及其在低背景噪声下检测目标序列的能力。
