Antithrombin aptamer

Another method for the analysis of aptamer-protein complexes involved the use of a positively charged ferrocene-tethered polythiophene, (19), as redox label reporting unit (Fig. 12.19). The antithrombin aptamer was immobilized on an electrode surface, and the electrostatic binding of the redox polymer (19) to the aptamer monolayer resulted in a supramolecular complex that revealed electrical contact between the polymer and the electrode.74 The formation of the aptamer-thrombin complex removed the polymer from the surface and blocked the electrical contact between the polymer label and the electrode. As a result, higher concentrations of thrombin increased the surface coverage of the aptamer-thrombin complex on the electrode, and this decreased the amperometric responses of the sensing device. 

Fig. 14 Schematic diagram for the principle of the developed ECL aptasensor for detecting thrombin. (A) The adsorption of thiolated antithrombin aptamer on and the 2-mercapto-ethanol block to the electrode. (B) The formation of the dsDNA between aptamer and its complementary ssDNA. (C) The intercalation of Ru(phen)3 into the dsDNA sequence. (D) Dissociation of dsDNA and release of Ru(phen)3 due to the interaction between thrombin and its aptamer. Reprinted with permission from Ref 97. Copyright (2009) American Chemical Society.

Neverthdess, what remained unknown at the outset generating modified DNA for in vitro sdection was whether modified DNAs could be recopied into unmodified DNA of complementary sequence in order to be ultimately doned thereby satisfying the third criterion for use in in vitro sdection. In 1994, Latham et al selected for antithrombin aptamers using 5-(l-pentynyl)-dUTP instead of dUTP, thus demonstrating that a modified nudeoside would be compatible with in vitro seleaion vide infra). In 1995, Eaton and co-workers described a portfolio of many other modified RNAs that contained different hydrophobic and amino modifications. 

The formation of aptamer-substrate complexes was also followed by the use of redox-active intercalators73 (Fig. 12.18d). A nucleic acid hairpin structure that contained in its single-stranded loop the antithrombin base sequence was assembled on a Au electrode, and methylene blue was intercalated as a redox label in the double-stranded stem of the hairpin structure. The hairpin was, then, opened in the presence of thrombin, by generating the respective G-quadruplex-thrombin complex, and as a result, the redox label was removed from the nucleic structure, showing a decrease in the voltammetric response with the increase in the concentration of thrombin. This method enabled the analysis of thrombin with a detection limit that corresponded to... 

Another label-free optical detection method—FTIR-ATR—has been applied for detection of thrombin by means of DNA aptamers [73], The antithrombin DNA aptamer previously developed by Tasset et al. [17] was immobilized covalently onto Si surface using UV irradiation method. As a quantitative measure, the area of N-H and CH2 bands was used. This method allowed to detect thrombin with a sensitivity around 10 nmol/L. The specificity of binding of protein to aptamer was also investigated using DNA with no binding site for thrombin. It has been noted that for effective binding study by FTIR-ATR method, the concentration of protein should be kept lower than 100 nmol/L. 

Holland, CA., Henry, A.T., Whinna, H.C., Church, F.C. (2000). Effect of oligodeoxy-nucleotide thrombin aptamer on thrombin inhibition by heparin cofactor II and antithrombin. FEBS Lett. 484, 87-91... 

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