## 3 Results and Discussion
### Construction and Evaluation of BRET-Based Biosensor with DNA-Protein Conjugates  
DNA aptamers are well known molecular binders with specificity to a specific molecule. In the present study, thrombin- and lysozyme-binding DNA aptamers were applied for the construction of a BRET-based biosensor with DNA-protein conjugates. For conjugation of DNA aptamer to the fusion protein via Rep, ssDNA comprised of the DNA aptamer and a Rep recognition sequence were reacted with the fusion protein. Lysozyme-binding DNA aptamer was first conjugated to VtpRNH. After conjugation, the emission spectra of lysozyme binding DNA aptamer-protein conjugates with or without lysozyme were evaluated. As shown in insets of Figure 4A,B, the emission spectra were normalized to the peak emission of NanoLuc at 450 nm, which was set to an intensity of 1.00. In the presence of BSA (100 uM), the normalized emission spectra of both DNA-protein conjugates with or without BSA were similar (Figure 4A). On the other hand, presence of lysozyme, the normalized emission peaks at 528 nm were found to increase compared to the control without lysozyme (Figure 4B). These results suggest that BRET efficiencies change upon binding of lysozyme to the DNA aptamers. To confirm this speculation, we evaluated the emission spectra in the presence of different concentrations of lysozyme. As shown in Figure 4C, the changes in BRET ratio (\(\Delta\)BRET) increased with increasing lysozyme concentration. These results confirm that the lysozyme-binding DNA aptamer-VtpRNH conjugate can be used as a BRET-based sensor. The \(K_{d}\) of the lysozyme-binding DNA aptamer is reported to be 30 nM [19]. Compared to this value, the sensitivity of our BRET-based biosensor with the lysozyme-binding DNA aptamer-VtpRNH conjugate is very low. As shown in Figure 3B, there were unreacted DNA. The sensitivity of sensor should be reduced by those unreacted DNA because unreacted DNA bind to target molecule competitively. However, in this case, it is not main reason for the low sensitivity. It was supposed that lysozyme formed aggregate in the presence of unreacted DNA caused by their net charge. Though monomeric lysozyme could not induce conformational change of fusion protein, BRET ratios would be changed by the aggregated lysozyme. The lysozyme binding DNA aptamer-VpRNH conjugate also showed similar results (Figure S1). We expected \(\Delta\)BRET to be larger for the lysozyme-binding DNA aptamer-VtpRNH conjugate compared with the DNA-VpRNH conjugate because tpRep was truncated to remove the flexible amino acid sequences from the N- and C-terminals. In contrast, the change in BRET ratio for the DNA-VtpRNH conjugate was smaller than that for the DNA-VpRNH conjugate. In case of DNA-VpRNH with high concentration of lysozyme, it was speculated that BRET ratio was changed not only by changes of the orientation but also the distance. Figure 3: Evaluation of DNA binding ability of the fusion protein. Design of the constructed fusion protein, VtpRNH (MW 57,900) **(A)**. DNA binding of tpRep in the fusion protein; the concentration of VtpRNH was 5 uM; white triangle indicates pRep without DNA conjugation and black triangle indicates pRep with DNA conjugation **(B)**. DNA-conjugation efficiency of protein was evaluated by ImageJ **(C)**. Emission spectrum of Venus at an excitation wavelength of 500 nm; the concentration of VtpRNH was 3 uM **(D)**. NanoLuc activities of fusion proteins. The emission spectrum was normalized to the peak emission of NanoLuc at 450 nm; final concentration of VtpRNH was 0.5 uM **(E)**. We also evaluated the thrombin-biding DNA aptamer conjugated to VtpRNH for use as a BRET-based biosensor. As shown in insets of Figure 5A,B, the normalized emission spectra of thrombin-binding DNA aptamer- and lysozyme-binding DNA aptamer-VtpRNH conjugates were evaluated in the presence of different thrombin concentrations. As shown in Figure 5A, the emission spectra of the lysozyme-binding DNA aptamer-VtpRNH conjugate were unchanged in the presence of different thrombin concentrations.