Rational Design of Fluorogenic and Spontaneously Blinking Labels for Super-Resolution Imaging

Abstract

Rhodamine dyes exist in equilibrium between a fluorescent zwitterion and a nonfluorescent lactone. Tuning this equilibrium toward the nonfluorescent lactone form can improve cell-permeability and allow creation of “fluorogenic” compounds—ligands that shift to the fluorescent zwitterion upon binding a biomolecular target. An archetype fluorogenic dye is the far-red tetramethyl-Si-rhodamine (SiR), which has been used to create exceptionally useful labels for advanced microscopy. Here, we develop a quantitative framework for the development of new fluorogenic dyes, determining that the lactone–zwitterion equilibrium constant (KL–Z) is sufficient to predict fluorogenicity. This rubric emerged from our analysis of known fluorophores and yielded new fluorescent and fluorogenic labels with improved performance in cellular imaging experiments. We then designed a novel fluorophore—Janelia Fluor 526 (JF526)—with SiR-like properties but shorter fluorescence excitation and emission wavelengths. JF526 is a versatile scaffold for fluorogenic probes including ligands for self-labeling tags, stains for endogenous structures, and spontaneously blinking labels for super-resolution immunofluorescence. JF526 constitutes a new label for advanced microscopy experiments, and our quantitative framework will enable the rational design of other fluorogenic probes for bioimaging.

Optimization and functionalization of red-shifted rhodamine dyes

“…1a). Our previous work on rhodamine dyes [3][4][5][6] allows definition of a general rubric that directly correlates the lactone-zwitterion equilibrium constant (KL-Z) of the free dye to the cellular performance of HaloTag 7 ligand derivatives ( Fig. 1b).…”

“…Rhodamines with intermediate KL-Z values exhibit improved cell and tissue permeability due to the modestly higher propensity of the molecule to adopt the lipophilic lactone form and rapidly traverse biological membranes 4,6 . Dyes exhibiting even smaller KL-Z values (KL-Z = 10 -2 -10 -3 ) preferentially adopt the closed lactone form, which can be exploited to create 'fluorogenic' ligands 6,9,10 as binding to the HaloTag protein shifts the equilibrium to the fluorescent form. This property also decreases in vivo utility, however, due to problems with solubility and sequestration in membranes.…”

“…Due to the inverse correlation of KL-Z and λmax, short-wavelength dyes would typically require tuning KL-Z lower to improve tissue permeability and create fluorogenic HaloTag ligands ( Fig. 1d) 4,6,10 . In previous work 4 , we found incorporation of 3,3-difluoroazetidines into the Janelia Fluor structure decreases KL-Z and elicits a hypsochromic shift of ~25 nm.…”

“…For the far-red and NIR rhodamines (5)(6)(7)(8)Fig . 1c) the KL-Z vs. λmax relationship reveals the need for alternative tuning strategy to increase KL-Z (Fig.…”

“…Although we have focused here on HaloTag ligands and mammalian cells, we expect this general rubric relating KL-Z to cellular performance ( Fig. 1b) to be applicable to other ligands and biological systems 6 . We also anticipate this expanded fluorophore palette to enable the rational design of finely tuned labels for biological imaging experiments in cells or animals and the new derivatization chemistry to facilitate the synthesis of novel ligands, labels, stains, and indicators, particularly those excited by far-red or NIR light.…”