Development of Fluorogenic Probes for Rapid High‐Contrast Imaging of Transient Nuclear Localization of Sirtuin 3

Abstract

Protein labeling using fluorogenic probes enables the facile visualization of proteins of interest. Herein, we report new fluorogenic probes consisting of a rationally designed coumarin ligand for the live‐cell fluorogenic labeling of the photoactive yellow protein (PYP)‐tag. On the basis of the photochemical mechanisms of coumarin and the probe–tag interactions, we introduced a hydroxy group into an environment‐sensitive coumarin ligand to modulate its spectroscopic properties and increase the labeling reaction rate. The resulting probe had a higher labeling reaction rate constant and a greater fluorescence OFF–ON ratio than any previously developed PYP‐tag labeling probe. The probe enabled the fluorogenic labeling of intracellular proteins within minutes. Furthermore, we used our probe to investigate the localization of sirtuin 3 (SIRT3), a mitochondrial deacetylase. Although the nuclear localization of SIRT3 has been controversial, this transient nuclear localization was clearly captured by the rapid, high‐contrast imaging enabled by our probe.

An expanded palette of fluorogenic HaloTag probes with enhanced contrast for targeted cellular imaging

“…Apart from Y-Halo, the reaction rates of the probes are slower than that of rhodamine-based HaloTag ligands but they are in the same range or faster than that observed for other reported solvatochromic HaloTag probes 16,18 and for alternative self-labeling protein tags such as SNAP-tag, CLIP-tag and PYP-tag. 4,5,10,12 After reaction with HaloTag, the spectral properties of the bound probes are essentially similar to those in glycerol but with lower fluorogenicities (Table 2). CCVJ-Halo and Y-Halo display comparable 12 to 15-fold fluorescence activation and while Red-Halo2-PEG and Orange-Halo perform slightly better, the four new probes have lower fluorescence enhancement factors than their parent compound Red-Halo2 (156-fold), which stems essentially from lower fluorescence quantum yields.…”

Illuminating Cellular Biochemistry: Fluorogenic Chemogenetic Biosensors for Biological Imaging

“…[83] Engineering of both fluorogens and PYP mutants led to enhanced brightness and labeling kinetics. [84][85][86][87] A sensor for methylated-DNA was obtained by fusing a methyl-CpG binding domain (MBD) with PYP. Labeling with an oxazole yellow (YO) ligand that strongly fluoresces upon DNA-binding allowed to track DNA-methylation during mitosis with good signal-to-noise ratio.…”

Engineering Glowing Chemogenetic Hybrids for Spying on Cells

“…[71] Introduction of a hydroxyl group into coumarin ligand enabled to increase the labeling reaction rate and the OFF-ON fluorescence ratio. [72] This probe allowed the observation of the transient nuclear localization of the mitochondrial deacetylase sirtuin-3.…”