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.

SUBMITTER: Yoshitomi T 

PROVIDER: S-EPMC8178692 | biostudies-literature |

REPOSITORIES: biostudies-literature