Structural and Binding Effects of Chemical Modifications on Thrombin Binding Aptamer (TBA)

Vibhav Valsangkar,Yicheng Huang,Goh Woon Lee,Waqas S Awan,Alan A Chen,Sweta Vangaveti,Walid M Fahssi,Jia Sheng

doi:10.3390/molecules26154620

Vibhav Valsangkar, Yicheng Huang + Show 6 more

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https://doi.org/10.3390/molecules26154620

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Abstract

The thrombin binding aptamer (TBA) is a promising nucleic acid-based anticoagulant. We studied the effects of chemical modifications, such as dendrimer Trebler and NHS carboxy group, on TBA with respect to its structures and thrombin binding affinity. The two dendrimer modifications were incorporated into the TBA at the 5′ end and the NHS carboxy group was added into the thymine residues in the thrombin binding site of the TBA G-quadruplex (at T4, T13 and both T4/T13) using solid phase oligonucleotide synthesis. Circular dichroism (CD) spectroscopy confirmed that all of these modified TBA variants fold into a stable G-quadruplex. The binding affinity of TBA variants with thrombin was measured by surface plasmon resonance (SPR). The binding patterns and equilibrium dissociation constants (KD) of the modified TBAs are very similar to that of the native TBA. Molecular dynamics simulations studies indicate that the additional interactions or stability enhancement introduced by the modifications are minimized either by the disruption of TBA–thrombin interactions or destabilization elsewhere in the aptamer, providing a rational explanation for our experimental data. Overall, this study identifies potential positions on the TBA that can be modified without adversely affecting its structure and thrombin binding preference, which could be useful in the design and development of more functional TBA analogues.

Highlights

Hemostasis is an important process for all living organisms
We further investigated the structural effects as well as the interactions of these modifications with thrombin through in silico modelling and molecular dynamics simulations studies, which indicate that the additional interactions or potentially stabilizing effects of the modifications are minimized either by the disruption of thrombin binding aptamer (TBA)–thrombin interactions or destabilization elsewhere in the aptamer, providing a rational explanation for our experimental observations
This study identifies potential positions on the TBA that can be modified without adversely affecting its structure and thrombin binding preference

Summary

Introduction

When a blood vessel is damaged, clotting factors are released and they convert prothrombin into thrombin [1]. Thrombin is a serine protease that plays a major role in the coagulation cascade [2]. It converts the soluble protein fibrinogen into the insoluble fibrin, which further stabilizes platelet aggregates and leads to clot formation. Anticoagulants are medications that help to prevent blood from over clotting. The current in-use anticoagulants, including warfarin, heparin and rivaroxaban [5,6], have several side effects, such as excessive bleeding, dizziness, hair loss, and tissue necrosis [7,8,9]. There is an urgent need for new anticoagulation therapies with enhanced efficacy and fewer side effects

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