Red‐Shifted Fluorogenic Substrate for Detection of lacZ‐Positive Cells in Living Tissue with Single‐Cell Resolution

Abstract

The Escherichia coli lacZ gene encoding b-galactosidase is awidely used reporter,but few synthetic substrates are available for detecting its activity with single-cell resolution in living samples.O ur recently reported fluorogenic substrate SPiDER-bGal is suitable for this purpose,b ut its hydrolysis product shows green fluorescence emission, and ar ed-shifted analogue is therefore required for use in combination with green fluorescent protein (GFP) markers.Herein, we describe the development of ar ed-shifted fluorogenic substrate for bgalactosidase,S PiDER-Red-bGal, based on as ilicon rhodol scaffold and ac arboxylic group as the intramolecular nucleophile.L acZ-positive cells were successfully labeled with SPiDER-Red-bGal at single-cell resolution in living samples, which enabled us to visualized ifferent cell types in combination with GFP markers.The Escherichia coli lacZg ene,w hich encodes b-galactosidase,i sapowerful reporter in combination with synthetic substrates, [1] and many lacZ-expressing animals have been established for experimental use.H owever,a lthough several substrates are available for detecting b-galactosidase activity, [2] selective labeling of lacZ-positive cells with single-cell resolution in living tissues or organisms is not feasible with most existing probes.W er ecently developed an ew class of functionalized fluorogenic substrate for b-galactosidase, SPiDER-bGal, [3] which is suitable for this purpose;w e controlled the fluorescence through intramolecular spirocyclization and achieved cellular retention by application of quinone methide chemistry.However,the utility of SPiDER-bGal is limited because it emits green fluorescence at around 550 nm, and therefore it is difficult to use in combination with green fluorescent protein (GFP), [4] one of the most widely used fluorescent tags for biological studies.A lthough red fluorescent proteins have been widely used as reporters in the red wavelength region, their tendency to form aggregates when expressed in neurons in mouse brain may limit their applicability. [5] Therefore,d evelopment of ar ed fluorescent probe for b-galactosidase with single-cell resolution should extend the utility of the lacZreporter.Herein, we describe the development of an ew fluorogenic substrate for b-galactosidase,w hich emits red fluorescence at above 600 nm without loss of the advantageous properties of SPiDER-bGal. We show that the new probe,SPiDER-Red-bGal, can label lacZpositive cells in combination with GFP markers and can successfully visualize multiple cell types at single-cell resolution in living samples.In order to develop red-shifted b-galactosidase probes,we first selected 2'Me-Sirhodol (Supporting Information, Scheme S1) as the core fluorophore,i nw hich the oxygen atom at the 10th position of rhodol is replaced with as ilicon atom;t his replacement has been reported to extend the absorption and fluorescence wavelengths by about 100 nm. [6] Indeed, 2'Me-Sirhodol seemed to have favorable properties for our purpose;i ts hows absorption/emission maxi...

First-generation species-selective chemical probes for fluorescence imaging of human senescence-associated β-galactosidase

“…In the eld of human b-gal recognition, E. coli b-gal, a bacterial enzyme encoded by lacZ, was routinely applied as the substitute enzyme of human b-gal in vitro, 26,27 as well as lacZ-transfected cells enriched by E. coli b-gal were commonly used as model organisms. [26][27][28][29][30][31] However, unlike human b-gal, bacterial b-gal is neither a lysosomal enzyme nor associated with senescence. 20,32,33 Although human b-gal shares a similar catalytic mechanism with E. coli b-gal, by comparing their catalytic domains, we found that this domain of human b-gal was very different from that of E. coli b-gal (pink for human b-gal, yellow for E. coli b-gal, RMSD ¼ 6.236Å), while it was very similar to that of its homologous Aspergillus oryzae (A. oryzae, a fungus) bgal (pink for human b-gal, cyan for A. oryzae b-gal, RMSD ¼ 1.002Å) (Fig.…”

A novel fluorescent off–on probe for the sensitive and selective detection of fluoride ions

“…Among the present analytical methods for F À , uorescent probes have caught great attention due to their high sensitivity and selectivity as well as easy operation. [11][12][13][14][15] To date, uorescent probes based on various sensing mechanisms have been reported for the detection of F À , including the uoride-hydrogen bonding interaction, 16,17 boron-uoride complexion 18,19 and uoride induced chemical reaction. [20][21][22][23][24][25][26][27][28][29][30][31][32] Among them the rst two types of probes could only work in non-aqueous media or media with high percentage of organic solvent because the detection signal would be largely hindered in aqueous environment.…”

Recent Progress in Small Spirocyclic, Xanthene-Based Fluorescent Probes

“…Hence, probes with a more red-shifted fluorescence are desired. We developed the red-emitting SPiDER-Red-βGal, which contains a 2 COOH-4CH 2 F-Sirhodol as the core fluorophore and a 2 -carboxy group as an intramolecular nucleophile to adapt the spirocyclic non-fluorescent form at physiological pH (Figure 2a) [102].…”

“…Hence, probes with a more red-shifted fluorescence are desired. We developed the red-emitting SPiDER-Red-βGal, which contains a 2′COOH-4CH2F-Sirhodol as the core fluorophore and a 2′-carboxy group as an intramolecular nucleophile to adapt the spirocyclic non-fluorescent form at physiological pH (Figure 2a) [102]. Upon cleavage of the βGal-moiety the reactive quinone methide can react with nucleophilic functional groups within the cell (e.g., cysteines or lysins of proteins) and hence be immobilized.…”