Intermolecular dark resonance energy transfer (DRET): upgrading fluorogenic DNA sensing

Abstract 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...

Full description

Saved in:

Bibliographic Details
Published in:	Nucleic acids research 2021-07, Vol.49 (12), p.e72-e72
Main Authors:	Barnoin, Guillaume, Shaya, Janah, Richert, Ludovic, Le, Hoang-Ngoan, Vincent, Steve, Guérineau, Vincent, Mély, Yves, Michel, Benoît Y, Burger, Alain
Format:	Article
Language:	eng
Subjects:	Chemical Sciences Methods Online
Online Access:	Get full text
Tags:	Add Tag No Tags, Be the first to tag this record!

Description
Summary:	Abstract 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.
ISSN:	0305-1048 1362-4962 1362-4962