Abstract
A new class of thrombin inhibitors based on sequence-specific single-stranded DNA oligonucleotides (thrombin aptamer) has recently been identified. The aptamer-binding site on thrombin was examined by a solid-phase plate binding assay and by chemical modification. Binding assay results demonstrated that the thrombin aptamer bound specifically to alpha-thrombin but not to gamma-thrombin and that hirudin competed with aptamer binding, suggesting that thrombin's anion-binding exosite was important for aptamer-thrombin interactions. To identify lysine residues of thrombin that participated in the binding of the thrombin aptamer, thrombin was modified with fluorescein 5'-isothiocyanate in the presence or absence of the thrombin aptamer, reduced, carboxymethylated, and digested with endoproteinase Arg-C. The digestion products were analyzed by reversed-phase high performance liquid chromatography and the peptide maps compared. Four peptides with significantly decreased modification in the presence of the aptamer were identified and subjected to N-terminal sequence analysis. Results indicated that B chain Lys-21 and Lys-65, both located within the anion-binding exosite, are situated within or in close proximity to the aptamer-binding site of human alpha-thrombin. The thrombin aptamer binds to the anion-binding exosite and inhibits thrombin's function by competing with exosite binding substrates fibrinogen and the platelet thrombin receptor.
Highlights
In this paper the area of thrombin involved in aptamer binding was investigated by both a solid-phase binding assay and by chemical modification of the thrombin-aptamer comitors are the first example of the use of this technique to plex
Competition of Thrombin Aptamer Binding to Immobilized a-Thrombin by y-Thrombin and Hirudin-To characterize the aptamer-thrombin interaction, asolid-phase plate binding assay was developedusing the biotinylated thrombin aptamer and a biotinylated 15-mer scramble sequence
Five hundred nanomolar biotinylated thrombin aptamer was preincubated for 1.5h a t room temperature with increasing concentrations of unbiotinylated thrombin aptamer (O), unbiotinylated 15-mer scramble sequence (A),a-thrombin (O),hirudin (m) or y-thrombin
Summary
The thrombin aptamer was shown to bind within the anion-binding exosite, and two of the lysine residues4 that Biotinylated thrombin aptamer or the biotinylated 15-mer scramble sequence were added at increasing concentrations and incubated for 2 h a t room temperature. 500 nM biotinylated thrombin aptamer was preincubated for 1.5 h a t room temperature with increasing concentrations of competitors.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.