Aptamers selection

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. 

A more recent application of redox labeled ODNs is redox-active aptamers that exploit molecular recognition between the aptamer and a target analyte. Briefly, aptamers are functional nucleic acids that selectively bind to a variety of targets. Due to a well-defined three-dimensional structure, aptamers can achieve selectivity comparable to that of antibodies but are readily accessible taking advantage of well-known nucleic acid chemistry, polymeric chain reaction and contemporary separation methods, followed by aptamer selection from random pools of nucleic acids (DNA or RNA) by in vitro selection process called systematic evolution of ligands by... 

As mentioned above, aptamers can also be made of DNA. Their selection (Figure 7.3) differs slightly from an RNA aptamer selection (Figure 7.2). Instead of the in vitro transcription of DNA into RNA, ssDNA is prepared as described in Section 7.3.3.1. Of course, inclusion of an RNA polymerase promoter in the template design, as well as the reverse transcription step, are not necessary. All other steps are the same as those for RNA aptamer selection (see Section 7.3.1). 

Bridonneau, P., Chang, Y.F., O Connell, D., Gill, S, C., Snyder, D.W., Johnson, L., Goodson, T. Jr., Herron, D.K. and Parma, D.H. (1998) High-affinity aptamers selectively inhibit human nonpancreatic secretory phospholipase A2 (hnps-PLA2), 3. Med. Chem. 41,118-786. 

Electrochemical methods of detection affinity interactions at the surfaces are rather effective due to their relative simplicity and low cost. Amperometric aptasensor based on sandwich assay was proposed by Ikebukuro et al. [42]. They used two aptamers selective to thrombin. 

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]).

The first aptasensor reported was particularly based on optical detection [66]. The 58-mer RNA aptamer selective to L-adenosine was immobilized onto the core of multimode fiber using avidin-biotin method. The detection was based on competitive binding of FITC-labeled L-adenosine with unlabeled analyte. This sensor also allowed to study the kinetics of binding and determine equilibrium constants. 

Andreola, M.L., Pileur, F., Calmels, C., Ventura, M., Tarrago-Litvak, L., Toulme, J.J. and Litvak, S. (2001) DNA aptamers selected against the HIV-IRNase H display in vitro antiviral activity. Biochemistry, 40, 10087-10094. 

Duconge, F. and Toulme, J.J. (1999) In vitro selection identifies key determinants for loop-loop interactions RNA aptamers selective for the TAR RNA element of HIV-1. RNA, 5, 1605-1614. 

Latham, J.A., Johnson, R. and Toole, J.J. (1994) The application of a modified nucleotide in aptamer selection novel thrombin aptamers containing 5-(l-pentynyl)-2l-deoyuridine. Nucl. Acids Res., 22, 2817-2822. 

Symensma, T.L., Giver, L., Zapp, M., Takle, G.B. and Ellington, A.D. (1996) RNA aptamer selected to bind human immunodeficiency virus type 1 are rev responsive in vitro. J. Virol., 70, 179-187. 

Toulme, J.-J. (2000) Aptamers selected oligonucleotides for therapy. Curr. Opin. Mol. Ther., 2, 318-324. 

An 18-mer DNA derived from in vitro selection to bind hemin has been previously reported. In a further report, the authors have prepared the RNA analogue of the aptamer to examine the peroxidative properties of each. Whilst both RNA and DNA aptamers displayed peroxidase activity, the RNA analogue bound with 30-fold weaker affinity. An RNA aptamer selected to inhibit the Drosophila B52 protein binds to B52 and inhibits B52-stimulated pre-mRNA splicing. It has been expressed in cell culture and animals, and binds B52 in vivo, suppressing all phenotypes. 

The solution structure of a DNA aptamer selected for binding to arginine revealed a hairpin loop with residues critical for binding in the loop. Binding arises from contact between the guanidino group and the phosphate backbone. The NMR characterisation of a kissing complex between an 18-nucleotide RNA... 

Table 1. Aptamers selected to bind small organic molecules.

Key words Proximity ligation, PLA, Aptamer, Selection, Real-time PCR, Detection. 

In the current method, we demonstrate how aptamers selected against antigens on the cell surface or against the cell surface itself can be adapted to PLA for the sensitive detection of small numbers of tumor cells (Fig. 1). 

A modified RNA aptamer, A9 [see (18)], that had been previously selected against the prostate-specific membrane antigen protein (PSMA) was used for the adaptation of aptamers to cell-mediated PLA. Aptamers have also been selected against whole cells and these can also be employed in PLA [see (10) for aptamer selection protocols]. 

DNA libraries A typical DNA library is composed of sequences containing a random-sequence region of 50-60 nucleotides (nt). The library is designated as LI, with the nucleotide sequence of 5 AGACCACAACGGTTTCCC-(N60)-TAGCATAACCCCTTG-3/, is used in some of our aptamer selection projects. 

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