Date of Award

1-1-2019

Language

English

Document Type

Master's Thesis

Degree Name

Master of Science (MS)

College/School/Department

Department of Chemistry

Content Description

1 online resource (iv, 28 pages) : illustrations (some color)

Dissertation/Thesis Chair

Jia Sheng

Committee Members

Mehmet Yigit, Ting Wang

Keywords

Binding study, Circular Dichroism (CD), Modified oligonucleotides, Surface Plasmon Resonance (SPR), Thrombin, Thrombin Binding Aptamer (TBA), Anticoagulants (Medicine), Blood coagulation factors, Quadruplex nucleic acids

Subject Categories

Chemistry

Abstract

The Thrombin Binding Aptamer (TBA), a short DNA strand with G-quadruplex conformation, binds to Thrombin with high affinity and acts as an inhibitor in the blood clotting. The TBA folds into a G-quadruplex conformation and stabilize the binding toward Thrombin. With this specificity of TBA with Thrombin, our goal is to enhance the binding affinity between them to observe the possibility of developing a new anticoagulant. First modification that we studied was dendrimer linkers which are called Trebler (T) and Long Trebler (LT). Those linkers were incorporated at the 5’ end of TBA by using solid phase oligonucleotide synthesis. Second type modification, NHS carboxy group was incorporated on thymidine within the active binding region of TBA sequence (T4, T13, and both T4, T13, respectively). Additionally, fluorescent group was also incorporated at the 5’ end of TBA with NHS carboxy modification on T4 an T13 positions. The binding affinity of modified TBAs to Thrombin was measured with Surface Plasmon Resonance (SPR) and the gel shift mobility assay was studied as well. Circular Dichroism (CD) spectroscopy allowed us to observe the overall stable G-quadruplex structure of the TBA variants. Both SPR and gel shift mobility assay, confirmed that all of these TBA variants we tested here in this thesis maintained the similar binding affinity as compared to the native counterpart in binding with Human ⍺ Thrombin.

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