Versatile naphthalimide tetrazines for fluorogenic bioorthogonal labelling

Graziotto, Marcus E.; Adair, Liam D.; Kaur, Amandeep; Vérité, Pauline M.; Ball, Sarah R.; Sunde, Margaret; Jacquemin, Denis; New, Elizabeth J.

doi:10.1039/d1cb00128k

Cited by 14 publications

(9 citation statements)

References 52 publications

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“…The kinetics traces at an incubation temperature of 60 °C, show the classical three-step profile, with the reaction reaching completion after only 3–4 h. The turbidity and ThT signals perfectly overlapped, suggesting that the aggregation reaction was entire of an amyloid-like origin 38 . Unlike previous studies 39 , 40 , we found fluorophore labeling had no influence on the aggregation kinetics (Supplementary Fig. 1b ).…”

Section: Resultscontrasting

confidence: 95%

Direct observation of heterogeneous formation of amyloid spherulites in real-time by super-resolution microscopy

et al. 2022

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Protein misfolding in the form of fibrils or spherulites is involved in a spectrum of pathological abnormalities. Our current understanding of protein aggregation mechanisms has primarily relied on the use of spectrometric methods to determine the average growth rates and diffraction-limited microscopes with low temporal resolution to observe the large-scale morphologies of intermediates. We developed a REal-time kinetics via binding and Photobleaching LOcalization Microscopy (REPLOM) super-resolution method to directly observe and quantify the existence and abundance of diverse aggregate morphologies of human insulin, below the diffraction limit and extract their heterogeneous growth kinetics. Our results revealed that even the growth of microscopically identical aggregates, e.g., amyloid spherulites, may follow distinct pathways. Specifically, spherulites do not exclusively grow isotropically but, surprisingly, may also grow anisotropically, following similar pathways as reported for minerals and polymers. Combining our technique with machine learning approaches, we associated growth rates to specific morphological transitions and provided energy barriers and the energy landscape at the level of single aggregate morphology. Our unifying framework for the detection and analysis of spherulite growth can be extended to other self-assembled systems characterized by a high degree of heterogeneity, disentangling the broad spectrum of diverse morphologies at the single-molecule level.

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Section: Resultscontrasting

confidence: 95%

Direct observation of heterogeneous formation of amyloid spherulites in real-time by super-resolution microscopy

et al. 2022

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“…18 However, some groups reported that the meta substituted isomers were shown to display higher turn-on ratios than the para derivatives. 28,29,39 Using TDDFT, we were also able to predict this distance on optimized structures (Supporting information, Figure S1). The ortho derivative was expected to be most effective for PeT, as it has the shortest distance between the two chromophores (7.6 Å), but all three regioisomers meet the distance criterion (<10 Å).…”

Section: Resultsmentioning

confidence: 99%

“…However, it was more unexpected for the TBET design. Several previous studies have reported that meta-connected tetrazine gives a higher turnon ratio than para isomers in TBET designs, [28][29]39 but the reasons for it have not been determined. A unique feature of our study is the comparison of ortho, meta and para substitutions on the phenyl spacer in the vinyl and ether series.…”

Section: Turn-on Ratio and Kineticsmentioning

confidence: 96%

Ultrabright two-photon excitable red-emissive fluorogenic probes for fast and wash-free bioorthogonal labeling in live cells

Auvray

Naud‐Martin

Fontaine

et al. 2023

Preprint

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Fluorogenic bioorthogonal reactions are promising tools for tracking small molecules or biomolecules in living organisms. Two-photon excitation, by shifting absorption towards the red, significantly increases the signal-to-noise ratio and decreases photodamages, while allowing to image about 10 times deeper than with a confocal. However, efficient two-photon excitable fluorogenic probes are currently lacking. We report here the design and synthesis of fluorogenic probes based on a two-photon excitable fluorophore and a tetrazine quenching moiety. Among our series of 6 new probes, our best candidate possesses a turn-on ratio of 22 and an impressive kinetic rate constant of 1.1x103M-1s-1, about 10 times higher than that reported for the fluorogenic iEDDA reaction with bicyclo[6.1.0]no-4-yn-9yl)methanol (BCN). Live-cell imaging under two-photon excitation confirmed that there was no need for washing to monitor the reaction between BCN and this probe with high-contrast images. The high two-photon brightness of the clicked adduct (362 GM) allows the use of a weak laser power compatible with in vivo imaging.

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“…In addition to their role as energy‐transfer quenchers, tetrazines can act as labeling groups with high specificity. If an alkene or alkyne is incorporated into the target, tetrazine will participate in an inverse electron demand Diels–Alder (IEDDA) reaction, [43] both fixing the fluorophore to the target, while uncaging the fluorophore to induce a turn‐on response [41a] . This makes tetrazine‐caged fluorophores potentially useful in SMLM techniques by giving a high contrast between the background and the labeled structures of interest (S/N).…”

Section: Photochemical Mechanisms Of Fluorophores Used In Smlmmentioning

confidence: 99%

Photochemical Mechanisms of Fluorophores Employed in Single‐Molecule Localization Microscopy

et al. 2022

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Decoding cellular processes requires visualization of the spatial distribution and dynamic interactions of biomolecules. It is therefore not surprising that innovations in imaging technologies have facilitated advances in biomedical research. The advent of super‐resolution imaging technologies has empowered biomedical researchers with the ability to answer long‐standing questions about cellular processes at an entirely new level. Fluorescent probes greatly enhance the specificity and resolution of super‐resolution imaging experiments. Here, we introduce key super‐resolution imaging technologies, with a brief discussion on single‐molecule localization microscopy (SMLM). We evaluate the chemistry and photochemical mechanisms of fluorescent probes employed in SMLM. This Review provides guidance on the identification and adoption of fluorescent probes in single molecule localization microscopy to inspire the design of next‐generation fluorescent probes amenable to single‐molecule imaging.

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Versatile naphthalimide tetrazines for fluorogenic bioorthogonal labelling

Abstract: New naphthalimide tetrazine probes permit fluorescent imaging of biomolecules in vitro and in living cells. They can be modified to provide previously unknown information about health and disease in biological systems.

Cited by 14 publications

References 52 publications

Direct observation of heterogeneous formation of amyloid spherulites in real-time by super-resolution microscopy

Direct observation of heterogeneous formation of amyloid spherulites in real-time by super-resolution microscopy

Ultrabright two-photon excitable red-emissive fluorogenic probes for fast and wash-free bioorthogonal labeling in live cells

Photochemical Mechanisms of Fluorophores Employed in Single‐Molecule Localization Microscopy

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