Validation and uncertainty analysis of ASTEC in early degradation phase against QUENCH-06 experiment

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.