Robust Evaluation of Intermolecular FRET Using a Large Stokes Shift Fluorophore as a Donor

Santana-Calvo, Carmen; Romero, Francisco Aguilar; López-González, Ignacio; Nishigaki, Takuya

doi:10.2144/btn-2018-0041

Cited by 9 publications

(6 citation statements)

References 21 publications

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“…An excellent review on large stokes shift dyes in such STED microscopy was published recently [29]. Another application of large stokes shift dyes is the Fӧrster resonance energy transfer (FRET) microscopy, a technique based on pair dyes that can undergo direct excitation of an acceptor by the adjacent donor molecules [30]. An effective dye pair needs overlapping in the emission and excitation spectra of donor and acceptor molecule, respectively, which inevitably leads to cross-excitation of the acceptor upon the donor, resulting in false-positive results.…”

Section: Introductionmentioning

confidence: 99%

Theoretical and Experimental Investigations of Large Stokes Shift Fluorophores Based on a Quinoline Scaffold

et al. 2020

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A series of novel styrylquinolines with the benzylidene imine moiety were synthesized and spectroscopically characterized for their applicability in cellular staining. The spectroscopic study revealed absorption in the ultraviolet–visible region (360–380 nm) and emission that covered the blue-green range of the light (above 500 nm). The fluorescence quantum yields were also determined, which amounted to 0.079 in the best-case scenario. The structural features that are behind these values are also discussed. An analysis of the spectroscopic properties and the theoretical calculations indicated the charge-transfer character of an emission, which was additionally evaluated using the Lippert–Mataga equation. Changes in geometry in the ground and excited states, which had a significant influence on the emission process, are also discussed. Additionally, the capability of the newly synthesized compounds for cellular staining was also investigated. These small molecules could effectively penetrate through the cellular membrane. Analyses of the images that were obtained with several of the tested styrylquinolines indicated their accumulation in organelles such as the mitochondria and the endoplasmic reticulum.

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Section: Introductionmentioning

confidence: 99%

Theoretical and Experimental Investigations of Large Stokes Shift Fluorophores Based on a Quinoline Scaffold

et al. 2020

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“…52 False-positive outcomes from overlapping in the emission and excitation spectra of the donor and acceptor molecule, respectively, can be resolved by integrating a large Stokes shi dye. 52,53 The provision of such integral would allow scientists to perform robust intermolecular FRET assays to investigate the molecular interactions. Styrylquinolines developed by Czaplińska and coworkers displayed a remarkable push-pull capability.…”

Section: Organic Moleculesmentioning

confidence: 99%

White light employing luminescent engineered large (mega) Stokes shift molecules: a review

et al. 2021

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“…A common concern about intermolecular FRET-based biosensors is the occurrence of background noise and false positives caused by over-production of the donor and acceptor fusions and cross-excitation of the acceptor by the excitation light of the donor. The problem of cross-excitation can be effectively solved by selecting a donor fluorophore with a large Stokes shift (75). The FRET donor we employed in this study, CouA, is a fluorophore with characteristic features ideal for FRET owing to its small size, high quantum field, and large Stokes shift (27).…”

Section: Discussionmentioning

confidence: 99%

FRET monitoring of transcription factor activities in living bacteria

Wang

Zhang

et al. 2022

Preprint

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Bacteria adapt to the constantly changing environments largely by transcriptional regulation through the activities of various transcription factors (TFs). However, techniques that monitor the in situ TF-promoter interactions in living bacteria are lacking. Herein, we developed a whole-cell TF-promoter binding assay based on the intermolecular Förster resonance energy transfer (FRET) between a fluorescent unnatural amino acid CouA which is genetically encoded into defined sites in TFs and the live cell fluorescent nucleic acid stain SYTO 9. We show that this new FRET pair monitors the intricate TF-promoter interactions elicited by various types of signal transduction systems with specificity and sensitivity. Furthermore, the assay is applicable to identify novel modulators of the regulatory systems of interest and monitor TF activities in bacteria colonized in C. elegans. In conclusion, we established a tractable and sensitive TF-promoter binding assay in living bacteria which not only complements currently available approaches for DNA-protein interactions but also provides novel opportunities for functional annotation of bacterial signal transduction systems and studies of the bacteria-host interface.

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Robust Evaluation of Intermolecular FRET Using a Large Stokes Shift Fluorophore as a Donor

Cited by 9 publications

References 21 publications

Theoretical and Experimental Investigations of Large Stokes Shift Fluorophores Based on a Quinoline Scaffold

Theoretical and Experimental Investigations of Large Stokes Shift Fluorophores Based on a Quinoline Scaffold

White light employing luminescent engineered large (mega) Stokes shift molecules: a review

FRET monitoring of transcription factor activities in living bacteria

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