Development of highly sensitive fluorescent probes for the detection of ß-galactosidase activity - application to the real-time monitoring of senescence in live cells.

We report the development of four novel fluorescent probes to monitor the activity of the ß-galactosidase enzyme (ß-gal), in vitro and in living cells. The fluorophores are based on a 6-amino-styryl-benzothiazole push-pull core and display a strong ICT emission. The probes encompass the fluorescent motif that is connected to a ß-d-galactopyranoside moiety through a self-immolative benzyl carbamate linker (ßGal-1-4). The screening of four different fluorophores enabled us to access new light-up and two-band ratiometric reporters. The four probes, ßGal-1-4, exhibited an extremely fast response and over 200-fold fluorescence enhancement (ßGal-1) following the enzymatic cleavage of the ß-d-galactopyranoside unit. This rapid and extremely sensitive response allowed the detection of senescence-associated ß-galactosidase (SA-ß-gal) activity; a widely used biomarker of senescence. More importantly, ßGal-1 also enabled us to monitor, in real-time, the emergence of senescence in live cells, i.e. the phenotypic transformation from normal to senescent cell. These findings underpin the fact that ßGal-1 may find useful applications in biomedical research. Importantly, ßGal-1 is suitable for epifluorescence and confocal microscopies, and flow cytometry techniques, which are among the most common analytical tools in biology.

Assessment of new triplet forming artificial nucleobases as RNA ligands directed towards HCV IRES IIId loop.

We report the synthesis of two new artificial nucleobase scaffolds, 1 and 2, featuring adequate hydrogen bonding donors and acceptors for the molecular recognition of U:A and C:G base pairs, respectively. The tethering of these structures to various amino acids and the assessment of these artificial nucleobase-amino acid conjugates as RNA ligands against a model of HCV IRES IIId domain are also reported. Compound 1e displayed the highest affinity (Kd twice lower than neomycin - control). Moreover, it appears that this interaction is enthalpically and entropically favored.