Design strategy of antidote sequence for bivalent aptamer: Rapid neutralization of high‐anticoagulant thrombin‐binding bivalent DNA aptamer‐linked M08 with HD22

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.

A neutralizable dimeric anti-thrombin aptamer with potent anticoagulant activity in mice

“…Furthermore, in vivo, the structural properties of the linker-connecting aptamers would also affect behavior; therefore, a classic flexible linker should also be investigated. Finally, we designed three M08s-1-based bivalent aptamers with M08s-dT 17 -M08s and M08s-dT 17 -TBA29 36 (Lin08-08 and Lin08-29) with the classic linear of 17 mer dT linker ( Figures 1 F and 1G), and M08s-ds 17 -M08s (Pse08-08) with a rigid linker with 17-bp duplex for further experiments ( Figure 1 H).…”

“…The experiment was performed based on a previous report. 36 Briefly, to a 0.6-mL tube, 100 μL of pre-annealed 12.5 nM aptamer in PBS and 100 μL of 6.25 nM human thrombin in PBS were added and incubated at room temperature for 15 min. 80 μL of the mixture was added to each well of a 96-well Costar® assay plate (Corning, WA, USA).…”