A DNA-scaffold platform enhances a multi-enzymatic cycling reaction
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We explored the co-localization of multiple enzymes on a DNA backbone via a DNA-binding protein, Gene-A* (A*-tag) to increase the efficiency of cascade enzymatic reactions.
Firefly luciferase (FLuc) and pyruvate orthophosphate dikinase (PPDK) were genetically fused with A*-tag and modified with single-stranded (ss) DNA via A*-tag. The components were assembled on ssDNA by hybridization, thereby enhancing the efficiency of the cascading bioluminescent reaction producing light emission from pyrophosphate. The activity of A*-tag in each enzyme was investigated with dye-labeled DNA. Co-localization of the enzymes via hybridization was examined using a gel shift assay. The multi-enzyme complex showed significant improvement in the overall efficiency of the cascading reaction in comparison to a mixture of free enzymes.
A*-tag is highly convenient for ssDNA modification of versatile enzymes, and it can be used for construction of functional DNA–enzyme complexes.
KeywordsA*-tag DNA scaffold Enzyme cycling reaction Firefly luciferase Multi-enzyme co-localization Pyruvate orthophosphate dikinase Proximity effect
This work was financially supported in part by Grants-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan. We thank Kikkoman Corporation for supplying BL assay reagents and PPDK gene.
Supplementary Table 1—DNA sequences in this study.