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

Abstract

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 DNA1). 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 recognition sequence and subjected to SDS-PAGE analysis. An ssDNA-dependent concentration band shift to higher molecular weight was observed for all DNA binding proteins upon covalent binding to each ssDNA. Based on the band quantification, we confirmed that UdgX had the highest binding efficiency and thus we used UdgX in following experiments.We attempted to detect Hb utilizing a biosensing element composed of GOx and anti-Hb aptamer fabricated using UdgX. We recently successfully integrated a protein coupling module, SpyCatcher/Tag system, into the universal fabrication method of sensing elements2-4). GOx and UdgX were also conjugated using SpyCatcher/SpyTag system, and then reacted with anti-Hb aptamer to fabricate aptamer-GOx complex. Hb has peroxidase activity and catalyzes chemiluminescence of luminol. Therefore, by using the aptamer-GOx complex, hydrogen peroxide produced by GOx is supplied to the Hb efficiently, and chemiluminescence of luminol is observed (Fig. 2).We confirmed homogeneous preparation of the aptamer-GOx complex by SDS-PAGE, and also the binding ability of aptamer and enzymatic activity of GOx. Using thus prepared DNA-protein complex, Hb was successfully detected with a high linearity in buffer and also in human serum without any washing procedures. The detection range was 4.0×10-3 to 32×10-3 g/L, this can meet the clinically required one (70 to 200 g/L) by diluting an actual sample. Because this system uses redox enzyme, it can be applied into the electrochemical measurement to achieve more rapid Hb detection. In this system, owing to integration of DNA binding protein and SpyCatcher/SpyTag system into sensing elements, a set of DNA aptamer and enzyme can be also easily exchanged, thus which has potential of various applications. References 1) P. B. Sang et al., Nucleic Acids Res., 43, 8452–8463 (2015).2) H. Kimura et al., Anal. Chem., 90, 14500-14506 (2018).3) H. Kimura et al., Biosens. Bioelectron., 175, 112885 (2021).4) D. Miura et al., Talanta, 234, 122638 (2021). Figure 1