Fluorescent DNA probes were prepared in a modular approach using the "click" post-synthetic modification strategy. The new glycol-based module and DNA building block place just two carbons between the phosphodiester bridges and anchor the dye by an additional alkyne group. This creates a stereocenter in the middle of this artificial nucleoside substitute. Both enantiomers and a variety of photostable cyanine-styryl dyes as well as thiazole orange derivatives were screened as "clicked" conjugates in different surrounding DNA sequences. The combination of the (S)-configured DNA anchor and the cyanylated cyanine-styryl dye shows the highest fluorescence light-up effect of 9.2 and a brightness of approximately 11,000 M-1 cm-1. This hybridization sensitivity and fluorescence readout were further developed utilizing electron transfer and energy transfer processes. The combination of the hybridization-sensitive DNA building block with the nucleotide of 5-nitroindole as an electron acceptor and a quencher increases the light-up effect to 20 with the DNA target and to 15 with the RNA target. The fluorescence readout could significantly be enhanced to values between 50 and 360 by the use of energy transfer to a second DNA probe with commercially available dyes, like Cy3.5, Cy5, and Atto590, as energy acceptors at the 5'-end. The latter binary probes shift the fluorescent readout from the range of 500-550 nm to the range of 610-670 nm. The optical properties make these fluorescent DNA probes potentially useful for RNA imaging. Due to the strong light-up effect, they will not require washing procedures and will thus be suitable for live-cell imaging.