Exploration of DNA Binding Proteins As a Versatile Tool for Fabrication of DNA-Protein Complexes and Its Application to Biosensing System

Introduction DNA-protein complexes composed of a DNA aptamer and an enzyme are useful to realize innovative biosensing systems; however, the stoichiometric and efficient fabrication method for DNA-protein complexes is yet to be developed. Chemical modification using amino acids on the surface of the protein is known as one irreversible conjugation way to fabricate DNA-protein complexes, but stoichiometric conjugation and retention of protein function to ensure high detection sensitivity are still a challenge. Therefore, in recent years, DNA binding proteins that covalently bind to specific DNA sequences have been utilized to fabricate DNA-protein complexes and applied as sensing elements. However, the binding efficiency to DNA of the DNA binding protein was low, which caused a decrease in detection sensitivity. Here, we introduce our challenge to utilize DNA binding proteins with high DNA binding ability as a versatile tool suitable for the fabrication of DNA-protein complexes. By fusing such DNA binding proteins to protein of interest, can be covalently linked to the fusion protein, leading to the preparation of stoichiometric DNA-protein complexes universally and conveniently (Fig. 1). We demonstrated the feasibility to construct a biosensing system of hemoglobin (Hb), composed of glucose oxidase (GOx) and anti-Hb aptamer as a representative example. Results & Discussion We explored the DNA binding proteins that can covalently bind to specific DNA sequences using the database to fabricate the stoichiometric and stable DNA-protein complex. Seventeen candidates were selected and classified into two families: HUH-endonuclease and uracil DNA glycosylase. We then selected three kinds of HUH-endonucleases: Rep domain of tomato yellow leaf curl virus (TYLCV), replication protein B (RepB), and conjugation transfer relaxase/helicase TraI (TraI). We expected them to have high recombinant production in the E. coli expression system because TYLCV and RepB have a relatively low molecular weight and TraI is derived from E. coli. We also selected UdgX because it is the only uracil DNA glycosylase known to form covalent bonds with DNA after removing uracil that aberrantly exists on DNA 1) . Thus, four DNA binding proteins with the ability to covalently bind to DNA were selected for further characterization. The binding ability of the four DNA binding proteins was compared. Each DNA binding protein was reacted with single stranded DNA (ssDNA) containing the corresponding