General Strategy To Improve the Photon Budget of Thiol-Conjugated Cyanine Dyes

Abstract

Maleimide-cysteine chemistry has been a routine practice for the site-specific labeling of fluorophores to proteins since the 1950s. This approach, however, cannot bring out the best photon budget of fluorophores. Here, we systematically measured the Cyanine3/5 dye conjugates via maleimide-thiol and amide linkages by counting the total emitted photons at the single-molecule level. While brightness and signal-to-noise ratios do not change significantly, dyes with thioether linkages exhibit more severe photobleaching than amide linkers. We then screened modern arylation-type bioconjugation strategies to alleviate this damage. Labeling thiols with phenyloxadiazole (POD) methyl sulfone, pchloronitrobenzene, and fluorobenzene probes gave rise to electron-deficient aryl thioethers, effectively increasing the total emitted photons by 1.5-3 fold. Among the linkers, POD maintains labeling efficiency and specificity that are comparable to maleimide. Such an increase has proved to be universal among bulk and single-molecule assays, with or without triplet-state quenchers and oxygen scavengers, and on conformationally unrestricted or restricted cyanines. We demonstrated that cyanine-POD conjugates are general and superior fluorophores for thiol labeling in single-molecule FRET measurements of biomolecular conformational dynamics and in two-color STED nanoscopy using site-selectively labeled nanobodies. This work sheds light on the photobleaching mechanism of cyanines under single-molecule imaging while highlighting the interplay between the protein microenvironment, bioconjugation chemistry, and fluorophore photochemistry.

Enhancing Gating Performance in Organic Molecular Field‐Effect Transistors by Introducing Polar Azulene Components

“…Among them, the thiol (À SH) group is one of the mostly used anchor groups. [23][24][25] The thiol group can self-assemble on gold surface by firstly desorbing the terminal hydrogen and then attaching to gold surface via a stable covalent SÀ Au bond. High electronic density in the thiol group enables a well electron transfer through the SÀ Au bond.…”

“…Therefore, anchor groups are frequently employed in constructing molecular devices. Among them, the thiol (−SH) group is one of the mostly used anchor groups [23–25] . The thiol group can self‐assemble on gold surface by firstly desorbing the terminal hydrogen and then attaching to gold surface via a stable covalent S−Au bond.…”

Live-Cell Mitochondrial Targeted NIR Fluorescent Covalent Labeling of Specific Proteins Using a Dual Localization Effect

“…Mammalian mitochondria encompass more than 1500 proteins, and perturbation of mitochondrial proteome causes neurodegenerative and metabolic disorders. − Most importantly, the large fluctuations in the local redox environment experienced by mitochondrial proteins, especially the thiol (SH) group of cysteine (Cys) residues, respond to these alterations through oxidative modifications. − Till now, approximately 1500 Cys moieties are identified from nearly 450 mitochondrial proteins, and it is a pretty good choice to label the exposed Cys-containing mitochondrial proteins. However, it is very arduous to target mitochondria due to the double-membrane architecture and highly negative inner mitochondrial membrane (IMM) potential [(ΔΨ m ) normal −150 to −180 mV]. − Cys-specific protein labeling to form protein-synthetic molecule hybrids is generally carried out with SH-reactive haloacetyl, disulfide, benzyl chloride, and maleimide moiety-containing fluorophores. − However, various studies indicate that thioether bond formation by the aforementioned approaches suffers a lack of preciseness in organelle-selective native protein labeling, and their instability in the live-cell microenvironment is of great concern. − …”

Revisiting molecularly conformation-planarized organic dyes for NIR-II fluorescence imaging

“…[36][37][38] A series of NIR-II fluorophores based on cyanine derivatives have been exploited to tune absorption/emission, aggregation behavior, and photoluminescence quantum yield (PLQY). [39][40][41][42][43] However, most of these dyes still have some unsatisfactory properties, like strong selfabsorption, structural instability, and low PLQY. 44 Important components of most promising contrast agent candidates are donor-acceptor-donor (D-A-D) type NIR-II dyes, which feature enhanced intermolecular charge transfer, tunable photophysical properties, and large Stokes shifts.…”