## 3 Results and Discussion
### Thermodynamic Stabilization and Binding Energy Distributions  
Based on the predicted binding structures (Supplementary Materials PDBs S1 and S3) of each DNA, i.e., the BAX sequence, and the p53-binding sequence from FTD-resistant cells, we predicted the binding structures (Supplementary Materials PDBs S2 and S4) of the BAX sequence incorporating FTD and the p53-binding sequence from resistant cells incorporating FTD. For these complexes, the conformational structures were sampled using the MD method at a biological temperature (310 K [-37 \({}^{\circ}\)C]) after equilibration for a sufficient time. Figure 4a,b shows the RMSDs of the DNA positions calculated for these sampling structures after superposing the p53 protein structures for BAX and binding sequences from FTD-resistant cells, respectively. The variation for a normal DNA and an FTD-incorporated DNA are shown as blue and orange lines, respectively. In the BAX sequence, FTD-incorporated DNA was found to have lower thermal oscillations than normal DNA, while normal DNA from FTD-resistant cells exhibited increased thermal stability compared to FTD-incorporated DNA. Due to these differences in thermal stability,  
Figure 2: Structural features of trifluoro-thymidine (FTD), and test sequences binding to p53. (**A**) The structural formula of FTD and (**B**) examples of adjacent bases that stabilize FTD. The presence of an electron-rich structure in the adjacent bases stabilizes the coordination through an attractive interaction with the trifluoromethyl group of FTD. The BAX response element sequences of (**C**) normal and (**D**) FTD-incorporated DNA. shown in Figure 5a,b, the difference in the binding energy distributions were fitted to the Gaussian-type function and that of box plots. In these figures, the blue and orange lines show the distribution of a normal and FTD-incorporated DNA, respectively. The p-values using \(t\)-test between the two distributions are 3.04 \(\times\) 10\({}^{-26}\) and \(\sim\)0, respectively. In these thermal samplings, the average relative distance between each DNA and the p53 is shown in Figure 6. The red circle indicates the position of FTD. Red- and blue-dashed circles indicate the average position of the FTD incorporated and the normal DNA loop position, respectively. Figure 4: Thermal vibration of each DNA in complex structures. The root mean square deviation (RMSD) analyses of the (**A**) BAX response element sequences and (**B**) p53-binding sequences extracted from trifluoro-thymidine (FTD)-resistant cells. The variation for a normal and FTD-incorporated DNA is shown as blue and orange lines, respectively. Figure 3: Validation using p53-binding DNA extracted from trifluoro-thymidine (FTD)-resistant cells. (**A**) The resistant strains were disrupted and p53-binding sequences were extracted. The sequences of (**B**) normal and (**C**) FTD-incorporated DNA used for verification. In our study, the binding energies were calculated using MM with the Amber force field, which represents qualitative rather than quantitative properties. As the probability of DNA recognition by p53 has been reported to be very low [25], the difference in the binding energy caused by sequence differences is also expected to be insignificant. Moreover, we found a slight increase in binding energy caused by FTD-incorporated DNA. However, this indicates that FTD-incorporated DNA binds more strongly to DNA-binding proteins and has higher recognition than normal DNA. In contrast, in p53-binding sequences extracted from FTD-resistant cells, we found that the binding affinity of FTD-incorporated DNA was greatly reduced compared to that of normal DNA. Therefore, we suggest that the random substitution of thymidine in FTD-resistant cells might confer resistance to FTDs by occupying a position that prevents them from binding to DNA-binding proteins. Corroborating this result, we observed that the average relative distance between FTD-incorporated DNA and p53 protein was closer for the BAX promoter sequence than for  
Figure 5: Comparison of binding energies.