Fig. 1. General concept of the intermolecularly assembled BRET-beacon. The sensor is constructed by site-specifically conjugating a handle-oligonucleotide to NanoLuc and hybridizing it to an anti-handle sequence appended to the target-binding stem-loop structure. The stem-loop is modified with a synthetic acceptor fluorophore (Cy3) to facilitate the readout of target oligonucleotide binding by measuring BRET efficiencies. Upon the hybridization of the target oligonucleotide to the complementary stem-loop sequence a rigid double-helix is formed that disrupts the stem, hereby separating NanoLuc and the synthetic acceptor fluorophore, causing the BRET efficiency to decrease.