## 3 Results and Discussion
### Construction and Evaluation of BRET-Based Biosensor with DNA-Protein Conjugates
On the other hand, the emission spectra of thrombin-binding DNA aptamer-VtpRNH conjugate increased with increasing thrombin concentration (Figure 5B). These results were also confirmed by calculation of \(\Delta\)BRET for each of the conjugates (Figure 5C). These results suggest that the BRET ratio changes only occurred with the appropriate combination of DNA aptamer and target molecules. Finally, to demonstrate specificity of the conjugates, we evaluated the BRET ratio of the thrombin-binding DNA aptamer-VtpRNH conjugate in the presence of more different concentrations of thrombin or BSA. As shown in Figure 6, \(\Delta\)BRET of the thrombin-binding DNA aptamer-VtpRNH conjugate increased with increasing thrombin concentration. On the other hand, \(\Delta\)BRET did not increase in the presence of different BSA concentrations. It is suggested that the \(\Delta\)BRET depends on the specific binding between DNA aptamers and target molecules. These results further suggest that the thrombin-binding DNA aptamer-VtpRNH conjugate can be used as a BRET-based thrombin biosensor. The \(K_{d}\) of the thrombin-binding DNA aptamer is reported to be -0.5 nM, which is different from the value obtained from our BRET-based biosensor with the thrombin-b  
Figure 4: Evaluation of lysozyme-binding DNA aptamer-protein conjugates with and without target molecules. Normalized emission spectra of lysozyme-binding DNA aptamer-VtpRNH conjugate in the presence of BSA (**A**) and lysozyme (**B**). Change in BRET ratio (\(\Delta\)BRET) as a function of lysozyme concentration (**C**). Figure 5: Evaluation of DNA aptamer-VtpRNH conjugate in the presence of different concentrations of thrombin. Conjugation with lysozyme-binding DNA aptamer (**A**) or thrombin-binding DNA aptamer (**B**). Change in BRET ratio (\(\Delta\)BRET) as a function of thrombin concentration (**C**). Conjugation with lysozyme-binding DNA aptamer (black diamonds) or thrombin-binding DNA aptamer (orange circles). conjugate [20]. One of the reasons of that is existence of unconjugated DNA. In this experiment, same amount of DNA aptamer and VtpRNH were mixed. From the result of Figure 3C, around half of DNA aptamer would remain as unreacted DNA. It should decrease the sensitivity of this sensor. To evaluate the property of BRET sensors, separations of DNA aptamer-VtpRNH conjugates are required. In addition, steric effects would reduce the sensitivity of the sensor. Steric effects are required to induce conformational changes. However, steric effects may also reduce the binding ability of the DNA aptamer. Therefore, the \(K_{d}\) of DNA aptamers should increase in our BRET-based biosensor. The square of the correlation coefficient (\(R^{2}\)) of the linear fit equation between concentrations of 200 and 1500 nM is close to 1 (\(R^{2}\) = 0.97). The square of the correlation coefficient was much closer to 1 compared to the results for the thrombin-binding DNA aptamer-VpRNH conjugate (\(R^{2}\) = 0.78) (Figure S2). These differences may be caused by the existence of flexible regions with the pRep protein, which could allow for movement of the BRET molecules even after binding of the target molecule to the DNA aptamer. These results suggest that DNA aptamer-VtpRNH conjugates exhibit the potential for use as BRET-based biosensors. While the sensitivity of these biosensors was too low compared with other DNA aptamer sensors, the sensitivity might be improved by changing the design of the fusion protein and DNA aptamers.