Fluorescent beacons based on silver (Ag) clusters for DNA/RNA detection represent a new type of turn-on probe that fluoresces upon hybridization to target nucleobase sequences. Physical-chemical mechanisms of their fluorescence activation still remain poorly understood. We studied in detail the fluorescence activation of dark Ag clusters induced by interactions of Ag-DNA complexes with different DNA sequences. In all cases, the final result depends neither on the location of the precursors (dark clusters) nor on their spectral properties. The reaction of fluorescence activation is a process similar to the growth of fluorescent silver clusters on dsDNA matrices. In both cases, reactants are dark clusters and two adjacent DNA strands. The latter form a double-stranded template for cluster nucleation. We found the optimized structure of a green fluorescent Ag4+2 cluster assembled on a C3/C3 DNA dimer in two different ssDNA pairs using QM modeling. The calculated absorption spectra match nicely the experimental ones, which proves the optimized structures. We conclude that apparent fluorescence activation in the studied systems results from reassembling Ag clusters on the new dsDNA template formed upon hybridization with the target. The suggested mechanism of "fluorescence activation"offers a way to design new light-up DNA probes. Two DNA strands making up the dsDNA template providing a high yield of bright Ag clusters can be used as the halves with the "stick"tails hybridizing with the base sequence of the target DNA. In this way, we have designed a light-up Ag cluster probe for β-actin mRNA.