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, p-chloronitrobenzene, 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.