Fig. 3. Characterization of the sequential assembly and target binding of the bioluminescent molecular beacon. (a) Normalized emission spectra of 10 nM ODN–NanoLuc (solid line), 10 nM ODN–NanoLuc hybridized to 20 nM stem-loop5 to form the bioluminescent molecular beacon (dotted line) and the bioluminescent molecular beacon incubated with 200 nM target oligonucleotide (dashed line). (b) Titration of various concentrations of stem-loop5 to 500 pM of ODN–NanoLuc. The stem-loop tightly binds with its anti-handle to the handle on ODN–NanoLuc as indicated by the linear increase in BRET-ratio up to stoichiometric concentrations of the stem-loop. Dotted lines represent linear fits of the experimental data from 0 pM to 500 pM and 500 pM to 3000 pM stem-loop5. (c) Titration of various concentrations of fully complementary target to 4 pM BRET-beacon5 (2 pM ODN–NanoLuc, 4 pM stem-loop5). An apparent affinity of K _d,app = 131.4 ± 8.9 pM is calculated by fitting of eqn (S1) (ESI†) to the experimentally obtained data. Triplicate measurements were performed in TE/Mg/NaCl buffer supplemented with 1 mg mL^–1 BSA (10 mM Tris–HCl, 1 mM EDTA, 12.5 mM MgCl₂, 150 mM NaCl, 1 mg mL^–1 BSA, pH 8.0, 1 : 5000 substrate).