Control of Thrombin Activity by Aptamer Modified Gold Nanoparticles

Autor: Chia-Lun Hsu, 許家綸
Rok vydání: 2011
Druh dokumentu: 學位論文 ; thesis
Popis: 99
We have demonstrated that incorporating sulfated galactose acid (sulf-Gal) into thrombin-binding-aptamer- (TBA-) conjugated gold nanoparticles (TBA–Au NPs) enables highly effective inhibition of thrombin activity toward fibrinogen. Au NP bioconjugates (TBA15/TBA29/sulf-Gal–Au NPs) were prepared from 13 nm Au NPs, 15-mer thrombin-binding aptamer (TBA15), 29-mer thrombin-binding aptamer (TBA29), and sulf-Gal. The numbers of TBA and sulf-Gal molecules per Au NP proved to have a strong impact on inhibitory potency. The best results were observed for 15-TBA15/TBA29/sulf-Gal–Au NPs (with 15 TBA15 and 15 TBA29 molecules per Au NP), which, because of their particularly flexible conformation and multivalency, exhibited ultrahigh binding affinity toward thrombin (Kd = 3.4 × 10−12 M) and thus extremely high anticoagulant (inhibitory) potency. Compared to the case without inhibitors (the “normal” value), their measured thrombin clotting time (TCT) was 91 times longer, whereas for TBA15 alone it was only 7.2 times longer. Their anticoagulant activity was suppressed by TBA-complementary-sequences- (cTBA-) modified Au NPs (cTBA15/cTBA29–Au NPs) at a rate that was 20 times faster than for free cTBA15/cTBA29. We also investigate the thymine-linker numer (Tn; n = 060) and hairpin number (hn = 016) of TBA impact the anticoagulant ability of TBA-Au NPs. The inhibitory potencies of TBA-Tn–Au NPs and TBA-hn–Au NPs to be in the following order: TBA-T30–Au NPs > TBA-T45–Au NPs  TBA-T60–Au NPs > TBA-T15–Au NPs > TBA-T0–Au NPs and TBA-h8–Au NPs > TBA-h16–Au NPs > TBA-h4–Au NPs > TBA-h0–Au NPs, respectively. We found the multivalent TBA15/TBA29-h8T15–Au NPs shown ultrahigh thrombin enzymatic inhibition in plasma samples. They exhibited a high binding affinity (Kd = 8.86 × 10−12 M) toward thrombin—over 100 times higher than that of monovalent TBA29, 10,000 times higher than that of TBA15, and at least 10 times higher than reported previously for fusion aptamers, dendritic aptamers, and TBA29–Au NPs. They exhibited TCT 296 times longer than for without inhibitor. We believe that our described techniques can be used widely to modify NPs with other anticoagulant DNA or RNA aptamers toward different proteins such as factor IX, activated protein C, and factor VIIa.
Databáze: Networked Digital Library of Theses & Dissertations
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