Biotechnology Letters

, Volume 40, Issue 4, pp 667–672 | Cite as

A DNA-scaffold platform enhances a multi-enzymatic cycling reaction

  • Yasumasa Mashimo
  • Masayasu Mie
  • Eiry Kobatake
Original Research Paper



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.


A*-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.

Supporting information

Supplementary Table 1—DNA sequences in this study.

Supplementary material

10529_2018_2517_MOESM1_ESM.docx (18 kb)
Supplementary material 1 (DOCX 17 kb)


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Copyright information

© Springer Science+Business Media B.V., part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Life Science and Technology, School of Life Science and TechnologyTokyo Institute of TechnologyYokohama-ShiJapan

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