![]() This method relies on preferential occupation of the protein active site by the inhibitor while the MB is in the closed conformation, thereby preventing enzymatic oxidation of the luminescent substrate. We hypothesized that a significant improvement in MB sensitivity can be achieved by eliminating unintended triggering of the bioluminescent reaction altogether. ![]() By designing a competitive inhibitor that is structurally very similar to the native substrate, essentially complete substrate exclusion can be realized. We hypothesized that another, more elegant, solution to inefficient quenching could be realized by capitalizing on the unique enzyme/substrate relationship of a specific bioluminescent protein. Although a drastic improvement in the signal amplitude for both systems was demonstrated when compared to fluorescence-based stem loop probes, similar drawbacks to a standard MB such as inefficient quenching of the bioluminescent signal were observed, resulting in a signal-to-noise ratio that could still be improved. Our laboratory also developed a BSLP using Gaussia luciferase (Gluc) rather than Rluc8 in order to improve sensitivity, as Gluc is smaller and has stronger bioluminescence activity. Modulation of the bioluminescent signal was essentially identical to a standard MB and was applied to the detection of miR-21, a commonly dysregulated biomarker. (13,14) For this, a bioluminescent protein ( Renilla luciferase, Rluc8) replaced the traditional fluorophore and demonstrated a significant improvement in sensitivity. (5) In order to address these shortcomings, our group was the first to report a bioluminescent stem-loop probe (BSLP). (11,12) However, they can suffer from severe sensitivity issues mainly due to background fluorescence from the excitation source. This type of fluorescent MB has been used in many applications besides nucleic acid detection, including real-time hybridization in living cells, (7,8) DNA–protein interactions, (9) monitoring enzymatic cleavage, (10) and real time polymerase chain reactions (qPCR). By conjugating a fluorophore/quencher pair to the stem termini, target-induced opening of the stem removes the quencher from close proximity of the fluorophore, leading to a dose-dependent increase in fluorescence. ![]() (6) Upon hybridization of a complementary target to the single-stranded loop region, the double-stranded stem is forced apart. Molecular beacon (MB) technology, first developed by Tyagi and Kramer in 1996, is a variant of the traditional hybridization-based fluorescence quenching assay platform in which the separate and independent donor/acceptor resonance energy transfer probes were replaced with a single-stranded oligonucleotide that self-hybridizes in a stem-loop configuration. We believe that this inhibitor approach may provide a simple alternative strategy to standard resonance quenching in the development of high-performance molecular beacon-based biosensing systems. To prove that a MAB can be more sensitive and have a better signal-to-noise ratio, a bioluminescence-based assay was developed against IFN-γ and provided an optimized, physiologically relevant detection limit of 1.0 nM. In this work, we designed a conjugated anti-interferon-γ (IFN-γ) molecular aptamer beacon (MAB) attached to a bioluminescent protein, Gaussia luciferase (GLuc), and an inhibitor molecule with a similar structure to the native substrate coelenterazine. A more elegant solution to inefficient quenching can be realized by designing a competitive inhibitor that is structurally very similar to the native substrate, resulting in essentially complete substrate exclusion. ![]() ![]() Although bioluminescent molecular beacons designed around resonance quenchers have shown higher signal-to-noise ratios and increased sensitivity compared with fluorescent beacon systems, bioluminescence quenching is still comparatively inefficient. ![]()
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