| Summary: | Abstract Background Bivalent thrombin‐binding aptamers (TBAs) have great potential for the treatment of thrombosis because they exhibit high anticoagulant activity, and their complementary single‐stranded DNA (ssDNA) sequences work as an antidote. However, a design strategy for antidote sequences against bivalent aptamers has not been established. Objectives To develop bivalent TBAs using M08, which exhibits higher anticoagulant activity than the previously reported exosite Ⅰ–binding DNA aptamers, such as HD1, an exosite Ⅱ–binding DNA aptamer (HD22) was linked to M08 with various types of linkers. In addition, short‐length complementary ssDNAs were designed to neutralize the optimized bivalent aptamer effectively and rapidly. Results Among the bivalent aptamers of M08 linked to HD22 with various types of linkers, M08‐T15‐HD22 possessed approximately 5‐fold higher anticoagulant activity than previously reported bivalent aptamers. To neutralize the activity of the 87‐meric M08‐T15‐HD22, complementary ssDNA sequences with different lengths and hybridization segments were designed. The complementary sequence against the M08 moiety played a more important role in neutralizing than that against the HD22 moiety. Hybridization of the T15 linker in the M08‐T15‐HD22 with the A15 sequence in the antidote accelerated neutralization due to toehold‐mediated strand displacement. Interestingly, some shorter‐length antidotes showed higher neutralizing activity than the full complementary 87‐meric antidote, and the shortest, 34‐meric antidote, neutralized most effectively. Conclusions A pair comprising an 87‐meric bivalent TBA containing M08 and a 34‐meric short‐length antidote with high anticoagulant and rapid neutralizing activities was developed. This design strategy of the DNA sequence can be used for other bivalent DNA aptamers and their antidotes.
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