Fig. 2. Synthesis and characterization of the semi-synthetic protein–DNA hybrid. (a) To obtain site-specific conjugation of the oligonucleotide handle to NanoLuc a 5′-amine-modified oligonucleotide is reacted with the heterobifunctional linker Sulfo-SMCC (i). Subsequently, the maleimide-activated oligonucleotide is reacted with a cysteine that is inserted in the C-terminus of NanoLuc (ii). (b) 4–20% SDS-PAGE analysis of the conjugation reaction of the oligonucleotide handle to NanoLuc and the final ODN–NanoLuc product. Conjugation efficiency based on relative band intensities of the crude reaction mixture is estimated to be >90%. Similarly, the purity of the final product is estimated to be >99%.