A rapid and concise setup for the fast screening of FRET pairs using bioorthogonalized fluorescent dyes

Abstract

One of the most popular means to follow interactions between bio(macro)molecules is Förster resonance energy transfer (FRET). There is large interest in widening the selection of fluorescent FRET pairs especially in the region of the red/far red range, where minimal autofluorescence is encountered. A set of bioorthogonally applicable fluorescent dyes, synthesized recently in our lab, were paired (Cy3T/Cy5T; Cy1A/Cy3T and Cy1A/CBRD1A) based on their spectral characteristics in order to test their potential in FRET applications. For fast elaboration of the selected pairs we have created a bioorthogonalized platform based on complementary 17-mer DNA oligomers. The cyclooctynylated strands were modified nearly quantitatively with the fluorophores via bioorthogonal chemistry steps, using azide- (Cy1; CBRD1) or tetrazine-modified (Cy3; Cy5) dyes. Reactions were followed by capillary electrophoresis using a method specifically developed for this project. FRET efficiencies of the fluorescent dye pairs were compared both in close proximity (5' and 3' matched) and at larger distance (5' and 5' matched). The specificity of FRET signals was further elaborated by denaturation and competition studies. Cy1A/Cy3T and Cy1A/CBRD1A introduced here as novel FRET pairs are highly recommended for FRET applications based on the significant changes in fluorescence intensities of the donor and acceptor peaks. Application of one of the FRET pairs was demonstrated in live cells, transfected with labeled oligos. Furthermore, the concise installation of the dyes allows for efficient fluorescence modification of any selected DNA strands as was demonstrated in the construction of Cy3T labeled oligomers, which were used in the FISH-based detection of Helicobacter pylori.

Beyond the Bioorthogonal Inverse-Electron-Demand Diels–Alder Reactions of Tetrazines: 2-Pyrone-Functionalized Fluorogenic Probes

“…All rights reserved. 7-(diethylamino) 2,3,8,9,cyclopropa [e][8]annulen-5-yl)ethynyl)-coumarin Accepted Manuscript This article is protected by copyright. All rights reserved.…”

“…This twoin-one feature of the tetrazine moiety was harnessed in the development of bioorthogonally applicable fluorogenic probes. 3 While the field of bioorthogonal chemistry and imaging probes have benefited greatly from tetrazines, the need for mutually orthogonal bioorthogonal reactions e.g., in multi-color labeling schemes called for the development of alternative bioorthogonal reactions. 4 This includes development of novel dienes for IEDDA schemes with substantially different reactivities toward strained alkenes/alkynes.…”

Tracking down protein–protein interactionsviaa FRET-system using site-specific thiol-labeling

“…First, we wanted to screen for FRET pairs from our azide and tetrazine derivatized dye-pool. [27][28][29][30] Since photophysical characteristics may substantially change upon conjugation to proteins, we needed a heterobifunctional tag that would enable Cys-specific targeting on the one hand and allow sequential one-pot high yielding modification both with azide-and tetra-zine-modified fluorescent probes. To this end we designed cyclooctyne bearing methylsulfonyl-oxadiazole 10 (Scheme 1).…”

Pixel-by-pixel autofluorescence corrected FRET in fluorescence microscopy improves accuracy for samples with spatially varied autofluorescence to signal ratio

“…Assuming a Poissonian distribution of detected photons per pixel with expected value λ, if systematic noise is negligible SNR is √λ, so lower intensities are expected to increase the uncertainty of the determined FRET E to a greater extent. Samples with low SNR can be made more amenable to FRET analysis by improving the quantum efficiency and photostability of the fluorescent dyes, by using more efficient detection 17 , optimizing the FRET dye pairs 18 , 19 , and by improved mathematical and statistical approaches 20 – 23 . Using the spectral spillover-corrected method generally allows for concluding about existing molecular interactions when a FRET efficiency of ~ 5% or above is seen.…”