Two-Factor Fluorogenic Cyanine-Styryl Dyes with Yellow and Red Fluorescence for Bioorthogonal Labelling of DNA.

An orange- and a red-emitting tetrazine-modified cyanine-styryl dyes were synthesized for bioorthogonal labelling of DNA by means of the Diels-Alder reaction with inverse electron demand. Both dyes use the concept of the "two-factor" fluorogenicity for nucleic acids: (i) The dyes are nucleic-acid sensitive by their non-covalent binding to DNA, and (ii) their covalently attached tetrazine moiety quench the fluorescence. As a result, the reaction with bicyclononyne- and spirohexene-modified DNA is significantly accelerated up to k2 =280,000 M-1 s-1 , and the fluorescence turn-on is enhanced up to 305. Both dyes are cell permeable even in low concentrations and undergo fluorogenic reactions with spirohexene-modified DNA in living HeLa cells. The fluorescence is enhanced in living cells to such an extent that washing procedures before cell imaging are not required. Their large Stokes shifts (up to 0.77 eV) also makes them well suited for imaging because the wavelength ranges for excitation and emission can be best possible separated. Furthermore, the spirohexene-modified nucleosides and DNA extend and improve the toolbox of already existing "clickable" dyes for live cell imaging.

Metabolic labelling of DNA in cells by means of the "photoclick" reaction triggered by visible light.

Two pyrene-tetrazole conjugates were synthesized as photoreactive chromophores that allow for the first time the combination of metabolic labelling of DNA in cells and subsequent bioorthogonal "photoclick" modification triggered by visible light. Two strained alkenes and three alkene-modified nucleosides were used as reactive counterparts and revealed no major differences in their "photoclick" reactivity. This is a significant advantage because it allows 5-vinyl-2'-deoxyuridine to be applied as the smallest possible alkene-modified nucleoside for metabolic labelling of DNA in cells. Both pyrene-tetrazole conjugates show fluorogenicity during the "photoclick" reactions, which is a second advantage for cellular imaging. Living HeLa cells were incubated with 5-vinyl-2'-deoxyuridine for 48 h to ensure one cell division. After fixation, the newly synthesized genomic DNA was successfully labelled by irradiation with visible light at 405 nm and 450 nm. This method is an attractive tool for the visualization of genomic DNA in cells with full spatiotemporal control by the use of visible light as a reaction trigger.

Fast and Efficient Postsynthetic DNA Labeling in Cells by Means of Strain-Promoted Sydnone-Alkyne Cycloadditions.

Sydnones are highly stable mesoionic 1,3-dipoles that react with cyclooctynes through strain-promoted sydnone-alkyne cycloaddition (SPSAC). Although sydnones have been shown to be valuable bioorthogonal chemical reporters for the labeling of proteins and complex glycans, nucleic acids have not yet been tagged by SPSAC. Evaluation of SPSAC kinetics with model substrates showed fast reactions with cyclooctyne probes (up to k=0.59 M-1 s-1 ), and two different sydnones were effectively incorporated into both 2'-deoxyuridines at position 5, and 7-deaza-2'-deoxyadenosines at position 7. These modified nucleosides were synthetically incorporated into single-stranded DNAs, which were successfully postsynthetically labeled with cyclooctyne probes both in vitro and in cells. These results show that sydnones are versatile bioorthogonal tags and have the premise to become essential tools for tracking DNA and potentially RNA in living cells.