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Aptamers Act as Activators for the Thrombin Mediated-Hydrolysis of Peptide Substrates

Authors

  • Dr. Xiaohong Tan,

    Corresponding author
    1. Department of Chemistry and Center for Nucleic Acids Science and Technology, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, Pennsylvania 15213 (USA)
    • Department of Chemistry and Center for Nucleic Acids Science and Technology, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, Pennsylvania 15213 (USA)

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  • Sourav Kumar Dey,

    1. Department of Chemistry and Center for Nucleic Acids Science and Technology, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, Pennsylvania 15213 (USA)
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  • Dr. Cheryl Telmer,

    1. Department of Biological Sciences, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, Pennsylvania 15213 (USA)
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  • Dr. Xiaoliang Zhang,

    1. Department of Chemistry and Center for Nucleic Acids Science and Technology, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, Pennsylvania 15213 (USA)
    2. School of Materials Science and Engineering, Beihang University, 37 Xue Yuan Road, Beijing 100191 (P.R. China)
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  • Prof. Bruce A. Armitage,

    Corresponding author
    1. Department of Chemistry and Center for Nucleic Acids Science and Technology, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, Pennsylvania 15213 (USA)
    2. Department of Biological Sciences, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, Pennsylvania 15213 (USA)
    • Department of Chemistry and Center for Nucleic Acids Science and Technology, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, Pennsylvania 15213 (USA)

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  • Prof. Marcel P. Bruchez

    Corresponding author
    1. Department of Chemistry and Center for Nucleic Acids Science and Technology, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, Pennsylvania 15213 (USA)
    2. Department of Biological Sciences, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, Pennsylvania 15213 (USA)
    • Department of Chemistry and Center for Nucleic Acids Science and Technology, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, Pennsylvania 15213 (USA)

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Abstract

Thrombin is the typical target in anticlotting therapy for many serious diseases such as heart attack and stroke. DNA aptamers are well-known thrombin inhibitors that prevent fibrinogen hydrolysis. We have discovered that exosite-targeting antithrombin aptamers enhance the activity of thrombin toward a small peptide substrate, Sar(N-methylglycine)-Pro-Arg-paranitroanilide, and that the activation of the enzyme by these aptamers is strongly inhibited by their complementary DNAs. Our study reveals that treatment with mixed aptamers or with a dual-aptamer construct led to an 8.6- or 7.8-fold enhancement in peptide hydrolysis relative to thrombin alone, a synergistic effect much higher than the activation observed with a monofunctional aptamer (1.5-fold for Apt27 or 2.7-fold for Apt15). In addition, we discovered that Apt27 is a biofunctional molecule for thrombin because of its activation effect. An enzyme kinetic study indicates that the binding of aptamers to exosites I and II significantly activates thrombin towards the peptide substrate, thus illustrating that binding of aptamers to exosites can allosterically regulate the active site of thrombin. Our study suggests the necessity of considering possible side effects when DNA aptamers are used for clinical applications involving the inhibition of thrombin-mediated clotting.

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