Construction of an Enzymatically-Conjugated DNA Aptamer–Protein Hybrid Molecule for Use as a BRET-Based Biosensor

DNA-protein conjugates are useful molecules for construction of biosensors. Herein, we report the development of an enzymatically-conjugated DNA aptamer–protein hybrid molecule for use as a bioluminescence resonance energy transfer (BRET)-based biosensor. DNA aptamers were enzymatically conjugated to a fusion protein via the catalytic domain of porcine circovirus type 2 replication initiation protein (PCV2 Rep) comprising residues 14–109 (tpRep), which was truncated from the full catalytic domain of PCV2 Rep comprising residues 1–116 by removing the flexible regions at the N- and C-terminals. For development of a BRET-based biosensor, we constructed a fusion protein in which tpRep was positioned between NanoLuc luciferase and a fluorescent protein and conjugated to single-stranded DNA aptamers that specifically bind to either thrombin or lysozyme. We demonstrated that the BRET ratios depended on the concentration of the target molecules.