Fig. 4. DNA-directed control of protein assembly via semisynthetic protein–DNA hybrids. (A) Split-EGFP complementation by DNA hybridization. Both biotinylated halves of split-EGFP are conjugated to two biotinylated oligonucleotides using streptavidin as crosslinker. Figure adapted from ref. 28. (B) DNA-directed complementation of split-mDHFR. Both protein halves are conjugated to a single-stranded oligonucleotide. Upon addition of a template strand that is complementary to the split-protein–DNA hybrids the protein halves colocalize and reassemble. Figure adapted from ref. 30. (C) DNA-templated assembly of a multi-enzyme complex. The biotinylated enzymes are conjugated to their biotinylated oligonucleotides using streptavidin as crosslinker. Efficient end product formation by the LUC luciferase is only observed upon colocalization of both enzymes on a shared template strand. NFOR reduces FMN to FMNH₂, which is subsequently consumed by the neighbouring LUC to convert a substrate to product under the emission of a photon. Figure adapted from ref. 31. (D) DNA-templated reassembly of the two subdomains of cytochrome P450 BM3. Using a DNA scaffold that contains a stem-loop structure, the overall efficiency of the enzyme-cascade can be reversible controlled by hybridization to a complementary effector oligonucleotide (adapted with permission from ref. 34, copyright 2011, American Chemical Society).