BRET-Based Biosensors for Nucleic Acid Detection and Impact of Molecular Crowding Monitoring

The advancement of biosensor technology and the demand for point-of-care diagnostics have driven the development of diverse methods for detecting viral infections and understanding complex biochemical processes. Nano luciferase (NLuc) bioluminescence has emerged as a promising tool in this field. Our research employs an advanced technique named Bioluminescence Resonance Energy Transfer (BRET) to design sensitive and robust biosensors. BRET depends on energy transfer from a luciferase protein (donor) to a fluorescent protein (acceptor) with compatible spectral overlap. We characterized the dynamic behavior of NLuc in both apo and furimazine-docked (holo) states through MD simulations. The mutational analysis highlighted that the R162A mutation led to a reduced yet stable bioluminescence activity of NLuc in vitro. Our work extended to designing BioNADs targeting SARS-CoV-2 variants by conjugating specific, fluorescently modified molecular beacons to NLuc-functionalized DNA for viral nucleic acid detection. Moreover, our study revealed that the decrease in BRET efficiency of BioNADs upon viral oligo binding could be visualized as a color change using a cell phone camera.

We observed the structural fluctuation of flexible linker compared to rigid linker. While flexible biosensor exhibited a faster decrease in enzymatic activity compared to rigid biosensor. However, both biosensors effectively monitored conformational changes of protein under crowded conditions. Our research anticipates NLuc’s broad utility in developing assays for prolonged bioluminescence monitoring. BRET-based biosensors are envisioned to revolutionize large-scale viral infection surveillance and enable monitoring of biochemical and biophysical changes within living cells, particularly in disease conditions characterized by intracellular molecular crowding.