DNA aptamer

Bruno J.G., Kiel J.L., In vitro selection of DNA aptamers to anthrax spores with electrochemiluminescence detection, Biosens. Bioelectron. 1999 14 457-464. 

Zhao W, Chiuman W, Lam JCF, McManus SA, Chen W, Cui Y, Pelton R, Brook MA, Li Y (2008) DNA aptamer folding on gold nanoparticles from colloid chemistry to biosensors. J Am Chem Soc 130(11) 3610-3618... 

Maehashi et al. (2007) used pyrene adsorption to make carbon nanotubes labeled with DNA aptamers and incorporated them into a field effect transistor constructed to produce a label-free biosensor. The biosensor could measure the concentration of IgE in samples down to 250 pM, as the antibody molecules bound to the aptamers on the nanotubes. Felekis and Tagmatarchis (2005) used a positively charged pyrene compound to prepare water-soluble SWNTs and then electrostatically adsorb porphyrin rings to study electron transfer interactions. Pyrene derivatives also have been used successfully to add a chromophore to carbon nanotubes using covalent coupling to an oxidized SWNT (Alvaro et al., 2004). In this case, the pyrene ring structure was not used to adsorb directly to the nanotube surface, but a side-chain functional group was used to link it covalently to modified SWNTs. 

Bera-Aberem M, Najari A, Ho HA, Gravel JF, Nobert P, Boudreau D, Leclerc M (2006) Protein detecting arrays based on cationic polythiophene-DNA aptamer complexes. Adv Mater 18 2703-2707... 

Recently, another example of a DNA-aptamer that was selected for binding to a small molecule and that was found to accelerate weakly a chemical transformation was reported [ 100]. These aptamers selected to bind to the fluorophor sulforhodamine B with high affinity were also capable of promoting the oxidation of a related molecule, dihydrotetramethyl rosamine, albeit with low efficiency. 

Blank M, Weinschenk T, Priemer M et al (2001) Systematic evolution of a DNA aptamer hinding to rat hrain tumor microvessels. Selective targeting of endothelial regulatory protein pigpen. J Biol Chem 276 16464-16468... 

Bayrac AT, Sefah K, Parekh P et al (2011) In vitro selecrion of DNA aptamers to glioblastoma multiforme. ACS Chem Neurosci 2 175-181... 

The detection of HIV-related proteins is one of the most challenging tasks. This is especially true because AIDS should be diagnosed as early as possible to enable an early and effective therapy of this infection. Pavski and Le (57) used the aptamer strategy to detect reverse transcriptase (RT) of the type 1 human immunodeficiency virus (HIV-1). A direct and specific ACE method was proposed using laser-induced fluorescence (ACE/LIF) as detection principle. Single-stranded DNA aptamers as probes fluorescently labeled were synthesized. The resulting aptamer is specific for HIV-1 RT, and it exhibited no cross-reactivity with RTs of the enhanced avian myeloblastosis virus (AMV), the Moloney murine leukemia virus (MMLV), or denatured HIV-1 RT. An affinity complex of RT 26-HIV-l RT was stable, with calibration curves linear up to 50 nM (6 /xg/mL) HIV-1 RT concentration. Both... 

The advantageous properties of PNA-based biosensors represent an appealing alternative to DNA and aptamer based sensors under some circumstances. They offer advantages of increased sensitivity, selectivity, and stability, therefore they present valuable alternative to DNA/aptamer based sensors but have problems associated with solubility and synthetic procedure. 

In this chapter, we introduce the essential steps that must be employed for the selection of (i) RNA aptamers, (ii) RNA aptamers modified at the 2 -position, and (iff) DNA aptamers, and we present experimental approaches to their realization. To cope with the variety of different selection problems, which depend strongly on the uniqueness of the chosen target and the intended application of the aptamer, we describe alternative procedures in some instances. 

Blank, M., Weinschenk, T., Priemer, M. and Schluesener, H. (2001) Systematic evolution of a DNA aptamer binding to rat brain tumor micro vessels, selective targeting of endo thelial regulatory protein pigpen, J. Biol. Chem. 276, 16464-16468. 

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... 

The results were explained by two effects (1) dehydration of cations and guanine 06 atomic group and (2) the water uptake upon folding of a single-stranded DNA into a G-quadruplex [29]. It is, however, interesting that DNA aptamer with high affinity to heparin binding site is insensitive to K+ ions [17]. 

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... 

MB has been shown useful also for detection of cocaine by means of specific DNA aptamer [50]. The MB-tagged aptamer has been immobilized via thiol group onto a gold support. In absence of cocaine, the aptamer was partially unfolded. Addition of cocaine resulted in folding of aptamer into three-way junction, moving MB to a close proximity with the electrode surface. This resulted in an increase in reduction peak measured by AC voltammetry. Sensor was regenerable and allowed to detect cocaine within several seconds with sensitivity below 10pmol/L. 

Label-free detection of ligand-aptamer interaction was also demonstrated by means of impedance spectroscopy technique [52,53]. Simultaneously, Radi et al. [52] and Rodriguez et al. [53] reported application of Faradaic impedance spectroscopy (FIS) in detection of interaction of proteins with DNA aptamers. The detection method is based on the measurement of resistance in presence of redox mediator Fe(CN)6-In absence of target protein, the negatively charged aptamer repulse the redox mediator molecules from the sensor surface. In a paper by... 

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... 

Yamana et al. [67] used bis-pyrene-labeled DNA aptamer for detection of ATP. The pyrene excimer was incorporated into several nucleotide positions. Addition of ATP resulted in an increase in fluorescence only for aptamers labeled by fluorescence probe between residues that were responsible for ATP binding. Using this sensor it was possible to detect ATP with mmol/L sensitivity. 

SPR method is currently well established and used in analysis of affinity interactions. The commercial instruments produced by Biacore AB or Texas Instruments are available in the market. Substantial progress in SPR instrumentation is connected also with the possibility to detect simultaneously the affinity interaction from several surfaces [72], This approach has been proved equally effective for detection of thrombin-DNA aptamer interactions [36]. 

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. 

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