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Novel DNA aptamer targeting VWF A1 domain
essary to bind the VWF A1 domain (Figure 6).
Due to the method limitations, the affinity between the VWF A1 domain and TAGX-0004 or ARC1779 was not analyzed by the SPR method. Since both oligonucleotides and the sensor chip are negatively charged, the KD value was underestimated when we immobilized the VWF A1 protein on the sensor chip and applied TAGX-0004 or ARC1779 as an analyte. Therefore, we performed alanine scanning by EMSA for DNA aptamers. In addition, the binding site of caplacizumab to VWF A1 domain was not
analyzed with EMSA, which was used to assess TAGX- 0004 and ARC1779, since this technique is not suitable for the analysis of proteins such as nanobodies and antibod- ies. Secondly, ARC1779 was originally PEGylated. However, we used ARC1779 without PEG in this study because we found no obvious differences between ARC1779 with and without PEG using RIPA, BIPA, T-TAS, and EMSA (data not shown).
In addition, caplacizumab was used in the clinical study and approved as a bivalent nanobody which has two
Figure 5. Analysis of caplacizumab binding sites to the von Willebrand factor A1 domain using alanine-scanning mutants with surface plasmon resonance. We performed surface plasmon resonance (SPR) analysis to investigate caplacizumab binding sites to the von Willebrand factor (VWF) A1 domain. After immobilizing caplacizumab on the sensor chip CM5, 16 alanine-substituted VWF A1 mutants were analyzed. The relative binding volume of wild-type (WT) VWF A1 was defined as 100%. The binding amount of each mutant was expressed as a ratio relative to WT. Five mutants were considered to bind to caplacizumab based on having a relative ratio above the cutoff of 80%. These results indicate that these five amino acids (K1362, R1392, R1395, R1399, and K1406) in the VWF A1 domain were important in caplacizumab binding.
Figure 6. Von Willebrand factor A1 domain binding sites to TAGX-0004, ARC1779, and caplacizumab. Amino acids colored yellow did not contribute to von Willebrand factor (VWF) A1 binding to aptamers or caplacizumab. Amino acids colored in red were necessary for binding to aptamers or caplacizumab. Three amino acids (F1366, R1395, and R1399) in the VWF A1 domain were identified as TAGX-0004 binding sites. Five amino acids (R1287, K1362, R1392, R1395, and R1399) were important for binding to ARC1779. Finally, five amino acids (K1362, R1392, R1395, R1399, and K1406) were important for binding to caplacizumab.
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