Juntit O.A.Yasamut U.Sakkhachornphop S.Chupradit K.Thongkum W.Srisawat C.Chokepaichitkool T.Kongtawelert P.Tayapiwatana C.Mahidol University2025-11-162025-11-162025-09-01Asian Pacific Journal of Allergy and Immunology Vol.43 No.3 (2025) , 737-7440125877Xhttps://repository.li.mahidol.ac.th/handle/123456789/112990Background: Assembly and budding in the late-stage of human immunodeficiency virus type 1 (HIV-1) production rely on Gag protein polymerization at the inner leaflet of the plasma membrane. We previously generated a monomeric ankyrin repeat protein (Ank1D4) that specifically interacts with capsid protein (CAp24) of HIV-1, however this protein had modest binding affinity. Objective: This study aimed to improve the avidity of Ank1D4 by generating two Ank1D4 dimers: (Ank1D4<inf>NC-NC</inf>) and its inverted form (Ank1D4<inf>NC-CN</inf>), with each domain connected by a flexible (G<inf>4</inf> S)<inf>4</inf> linker peptide. Methods: Binding properties of monomeric and dimeric Ank1D4 was performed by capture enzyme-linked immunosorbent assay (ELISA). Sandwich ELISA was used to examine bifunctional module of dimeric Ank1D4. Ank1D4<inf>NC-NC</inf> and Ank1D4<inf>NC-CN</inf> were evaluated using bio-layer interferometry (BLI), compared to monomeric Ank1D4. Results: Similar binding surfaces were observed in both dimers which was comparable with monomeric Ank1D4. The interaction of Ank1D4<inf>NC-CN</inf> with CAp24 was significantly greater than that of Ank1D4<inf>NC-NC</inf> and Ank1D4 by capture ELISA. Ank1D4<inf>NC-CN</inf> also exhibited bifunctionality using a sandwich ELISA. The KD of Ank1D4<inf>NC-CN</inf>, Ank1D4<inf>NC-NC</inf> and monomeric Ank1D4 was 3.5 nM, 53.7 nM, and 126.2 nM, respectively using bio-layer interferometry analysis. Conclusions: This study provides a strategy for increasing Ank1D4 avidity through the construction of novel inverted dimers with a flexible linker. Ank1D4<inf>NC-CN</inf> may provide an alternative treatment strategy for inhibiting HIV-1 replication.MedicineImmunology and MicrobiologyArticleSCOPUS10.12932/ap-210122-13102-s2.0-1050208263652228869435598193