Assembly of zinc finger motif-fused enzymes on a dsDNA scaffold for catalyzing consecutive reactions with a proximity effect

The feasibility of assembling enzymes, catalyzing consecutive reactions, on to a double-stranded DNA (dsDNA) scaffold utilizing zinc finger motifs is described. The catalytic activities of two zinc finger motif-fused enzymes catalyzing a bioluminescence reaction with energy recycling, namely pyruvate phosphate dikinase and firefly luciferase, have been evaluated. Bioluminescence measurements with dsDNA scaffolds coding a different distance between the binding sites for each zinc finger motif-fused enzyme confirmed the effect of the distance, proving the proximity effect of ATP recycling presumed to be the result of efficient intermediate diffusion. Thus, fusion to zinc finger motifs offers a promising option for the assembly of bi-enzymes, catalyzing a consecutive reaction, onto a dsDNA scaffold with a proximity effect.