Thrombin-binding DNA aptamer

Macaya RF, Sdiultze P, Smith FW, Roe JA, Feigon J Thrombin-binding DNA aptamer forms a unimolecular quadnqilex structure in solution. Proc Natl Acad Sci USA (1993) 90 3745-3749. 

P. Schultze, R.F. Macaya and J. Feigon, Three-dimensional solution structure of the thrombin-binding DNA aptamer d(GGTTGGTGTGG TTGG), J. Mol. Biol, 1994, 235, 1532-1547. 

Figure 11.3 MALDI-TOF-MS results for 50 pmol of thrombin incubated for 30 minutes at room temperature on (A) a thrombin-binding DNA aptamer-coated spot and on (B) a scrambled oligonucleotide-coated spot, followed by rinsing to remove unbound or loosely bound protein. [From Dick et al. (2004).]...

Macaya, R.F. et al.. Thrombin-binding DNA aptamer forms a unimolecular quadruplex structure in solution, Proc. Nad Acad. Sci. USA, 90,3745,1993. 

Electrochemical indicator methods are based on the application of redox probe that undergoes oxidation and reduction transition due to electron transfer from electrode surface to a probe. In 2005, several studies that used methylene blue (MB) as an electrochemical indicator were published. MB is positively charged low-molecular-weight compound that can be reduced by two electrons to a leucomethylene blue (LB). The reduction process can be effectively monitored, e.g., by differential pulse voltammetry or coulometry. In presence of redox probe Fe(CN)6, the LB is oxidized to MB and the system is regenerated [44,45]. In papers by Hianik et al. [31,46], MB was used as the indicator of detection of interaction of human thrombin with DNA aptamer. The method of detection is schematically shown in Fig. 33.3B. MB binds both to DNA and to the protein. For charge transfer from electrode to MB, i.e., for MB reduction, it is important that MB should be close to the electrode surface. Therefore, the charge transfer from the electrode... 

Polyanions snch as heparin bind to the thrombin and could affect the binding affinity of proteins to the aptamer. To verify this hypothesis, we studied the interaction of thrombin with DNA aptamer at molar ratios of thrombin to heparin 1 1, 1 3, and 1 10. The thrombin, heparin, or their complexes were added to the buffer stepwise from a stock solution, and the charge transfer was measnred... 

FIGURE 16.5 (a) Cocaine 13 binding DNA aptamer bearing fluorescein and dabcyl units at 5 and 3 ends. (b) MAB for thrombin 14 binding. (c) thrombin beacon design. (Sources Copyright (2005), American Chemical Society and copyright (2002), Elsevier. With permission.)... 

Fig. 33.1. Structure of DNA aptamers for recognition fibrinogen (A) [15,29] and heparin binding sites (B) [17] of thrombin. The non-canonical hydrogen bonds between guanines are shown by dotted lines. In the case of structure (A), the spacer composed of 15 T chain terminated by thiol group at the end of hydrophobic spacer is shown [34]. (C) Structure of RNA aptamer against fibrinogen binding site of thrombin [23].

Typical two-stacked guanine tetramers in the active binding site of the DNA aptamers against thrombin were typical also for other aptamers subsequently developed, including RNA aptamer (see, e.g., aptamer against cellular prion [25-27]). Apart from linear aptamers... 

Fig. 33.5. The plot of the series resonance frequency of AT-cut crystal covered by neutravidin and with immobilized biotinylated 32-mer DNA aptamer selective to heparin binding site of thrombin as a function of thrombin and HSA concentration, respectively. The fundamental frequency of the crystal was 9MHz. The frequency was determined by HP4395A Network-Spectrum analyzer (Hewlet Packard, Colorado Springs, CO, USA). The crystal was placed in a flow cell developed by Thompson et al. [37] (experiment was performed by I. Grman in M. Thompson laboratory [75]).

Low-wave acoustic sensor [63] was used to detect interaction of thrombin with RNA and DNA aptamers [24]. The authors compared the binding of thrombin to RNA aptamer also by using filter binding method utilizing radiolabeling of RNA aptamer by 3 -P32 and also by... 

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. 

Use, for example, 32-mer DNA aptamer modified by biotin at 3 end of the following sequence 3 -BIO-GGG TTT TCA CTT TTG TGG GTT GGA CGG GAT GG - 5. This aptamer has at its 5 end typical motif with high affinity to the heparin-binding site of the thrombin [1], (Please note that aptamer that selectively bind the fibrinogen-binding site of thrombin has a different structure—see Chapter 33 in CD accompanying this book). The aptamer can be purchased from Thermo Electron Corporation, Germany. 

Pabo sky LR, McCurdy SN, Griffin LC, Toole JJ, Leung LLK The single-stranded DNA aptamer binding-site of human thrombin. JBiol Chem (1993) 268 20808. 

Single-stranded DNA aptamers identified were found to bind thrombin with a value of 25-200 nM. The central core (15-17 residues in length) was found to assume a G-quartet structure, a very common structural motif in both DNA and RNA aptamers (Figure 10.15). DNA aptamers that bind other proteins have since been found to have several... 

Aptamer-based protein sensors have been developed since 1998. Thus, 0.7 amol of thrombin in 140-pL sample (0.5 pM concentration) was detected in a single-aptamer sensor through binding to a fluorescently labeled DNA aptamer by evanescent-wave-induced fluorescence anisotropy in less than 10 minutes [45]. Performance of DNA... 

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