## 1 Introduction  
In biomolecules, changes in partial structure, such as amino acid mutations in proteins [1, 2], methylation or acetylation of nucleic acids [3, 4, 5], can significantly alter their molecular functions. For example, even a single amino acid substitution in a protein sequence can significantly change the structure of the protein, and the biological function may differ accordingly [2]. In addition, it has been shown that the presence or absence of methylation in microRNAs can change the binding interaction with dicer proteins, resulting in differences in the binding rate to complementary RNAs [5]. Therefore, it is very important to observe subtle differences in structure at the single molecule level in biomolecules and to understand their functions. In recent years, it has become possible to experimentally observe subtle differences within a single molecule by using tunneling current-based sequencing [6, 7] and other techniques [8]. On the contrary, analysis based on theoretical calculations can predict how changes in substructure will affect intermolecular interactions before they are observed experimentally. Typical simulation calculations based on theoretical physics and chemistry, which are widely used in the life sciences, fall into three main categories. The three methods are the molecular docking method [9, 10], which is used to search for bonding conformations between biomolecules; the molecular dynamics (MD) method [11, 12], which is used to understand the thermodynamic behavior of molecules; and the molecular orbital (MO) method [13], which can precisely interpret molecular reactivity and intermolecular interactions at the electronic level. By effectively combining these methods, it has become possible to understand various in vivo environments. In particular, theoretical simulations are effective because they allow detailed analysis of how small conformational changes at the single-molecule level cause changes in interactions with the molecule of interest. However, it is extremely difficult to predict from scratch how the molecular interactions between DNA and its binding proteins, which have undergone minute structural changes, will change compared to the original nucleic acid, using current theoretical calculations. This is because it is difficult to predict the correct binding conformation of a protein that recognizes the double helical DNA structure by the conventional molecular docking method alone, because most of the interfaces that come into contact with DNA are deoxyribose portions. Therefore, we proposed a new strategy, BC-BEP (Binding Conformation and Binding Energy Prediction), to analyze how subtle conformational differences in nucleic acids alter the binding interaction and binding energy between DNA and binding proteins.  
BC-BEP uses DNA-protein complex structure data registered in the Protein data bank (PDB) to predict the complex structure of a DNA-binding protein and its target DNA based on homology at various binding conformations. The predicted complex structure is then used as the initial structure, and MD calculations are performed under conditions that mimic the in vivo environment to sample the thermodynamic behavior. These sampled conformations will be used to enable binding energy estimation. To show whether BC-BEP can explain differences in binding changes at the level of a single nucleic acid molecule incorporated into DNA, we tested it with trifluoro-thymidine (FTD) [14], an anticancer drug known to exert high antitumor effects when incorporated into DNA. The binding prediction strategy proposed in this study is strongly expected to be useful in clarifying in vivo characteristics, such as functional changes due to DNA methylation.