## 1 Introduction  
DNA-protein conjugates are useful materials for biosensing applications [1]. Use of DNA in these conjugate molecules allows for both signal amplification as well as molecular recognition (e.g., using DNA aptamers) [2; 3; 4; 5]. DNA aptamers bind to specific molecules with high affinity and specificity. Therefore, DNA aptamer-based biosensing systems have been constructed as conjugates of DNA aptamers and reporter molecules [6; 7; 8]. Single-stranded DNA (ssDNA) containing aptamer sequences are modified at the 5\({}^{\prime}\) and/or 3\({}^{\prime}\) ends with a fluorophore for use in construction of DNA aptamer-based biosensors [9; 10; 11; 12]. In addition to modification of ssDNA with a fluorophore, a reporter protein can also be conjugated to ssDNA [3; 4; 5]. However, conventional methods for conjugation of ssDNA to a reporter protein require cumbersome procedures. To overcome these limitations, we developed a method for conjugation of ssDNA to a protein of interest fused with a replication initiation protein (Rep) [5; 13]. In this method, a protein fused with Rep can be covalently linked to ssDNA via enzymatic reaction, without the need for any chemical modification of the ssDNA. Recently, the catalytic domain of porcine ciroovirus type 2 Rep comprising residues 1-116 (pRep) was employed to construct DNA-NanoLuc luciferase (NanoLuc) conjugates [5]. DNA-NanoLuc conjugates were applied for use in a DNA aptamer-sandwich assay system. Moreover, pRep retains its DNA binding activity regardless of whether the protein of interest is fused to the N-terminus or C-terminus. Bioluminescence resonance energy transfer (BRET)-based biosensors are frequently used in biosensing methods [14; 15; 16]. For the design of intramolecular BRET sensors, sensory domains that lead to conformational changes are located between donor and acceptor molecules, such as luciferase and a fluorescent protein. Therefore, we decided to design a fusion protein for application as a BRET biosensor, in which pRep was positioned between luciferase and a fluorescent protein, and then a DNA aptamer was conjugated via pRep. It was anticipated that our designed fusion protein conjugates containing DNA aptamers might undergo conformational changes upon binding of a target molecule to the DNA aptamer (Figure 1). BRET efficiency is dependent on not only the distance between the donor and acceptor molecules, but also their relative orientations. In our design, it was speculated that larger change in the distance between donor and acceptor molecules would not be occurred even when a target molecule binds to the DNA aptamer. However, the BRET efficiency was expected to change through alteration of their orientation upon binding of a target molecule to the DNA aptamer. Herein, we tested this idea by constructing a fusion protein in which pRep was positioned between Venus as an acceptor fluorophore and NanoLuc as an energy donor. Overlap of the bioluminescence emission spectrum with the excitation spectrum of the acceptor fluorophore is important for selection of a BRET molecule pair. The combination of Venus and NanoLuc has been previously applied in several BRET-based sensors [15; 16]. Energy transfer occurs when the donor and acceptor molecules are within 10 nm of each other in proximity [14; 17]. Based on the known structure of pRep, we expected that BRET between NanoLuc and Venus would occur in the designed fusion protein. As noted above, the BRET efficiency depends not only on the distance between the donor and acceptor molecules, but also their relative orientations. To overcome limitations associated with molecular orientation, linkers are generally inserted between the sensory domain and the BRET molecules to allow for a certain degree of movement of the BRET molecules [14]. pRep exhibits flexible regions at the N- and C-terminals [18]. In the present study, we expected the orientation change upon binding of target molecules to sensor.