Selection of Aptamers

Combinatorial Hbraries are limited by the number of sequences that can be synthesized. For example, a Hbrary consisting of one molecule each of a 60-nucleotide sequence randomized at each position, would have a mass of >10 g, weU beyond the capacity for synthesis and manipulation. Thus, even if nucleotide addition is random at all the steps during synthesis of the oligonucleotide only a minority of the sequences can be present in the output from a laboratory-scale chemical DNA synthesis reaction. In analyzing these random but incomplete Hbraries, the protocol is efficient enough to allow selection of aptamers of lowest dissociation constants (K ) from the mixture after a small number of repetitive selection and amplification cycles. Once a smaller population of oligonucleotides is amplified, the aptamer sequences can be used as the basis for constmcting a less complex Hbrary for further selection. 

Cox, J.C., Hayhurst, A., Hesselberth, J., Bayer, T.S., Georgiou, G., and Ellington, A.D., Automated selection of aptamers against protein targets translated in vitro from gene to aptamer. Nucleic Acid Res., 30(20), 1-14, 2002. 

Selection of Aptamers that Bind to the nAChR and Displace Cocaine... 

Aptamers are nucleic acids which exhibit a defined structure due to their nucleotide sequence and therefore, are able to specifically bind selected targets [1] (aptus [lat.] = fitting, sticking to). Aptamers and likewise, ribozymes [2] and deoxyribozymes [3] are selected in vitro by screening nucleic acid libraries. Here we describe in detail the selection of aptamers by a process called SELEX (Systematic Evolution of Ligands by Exponential enrichment) [4]. 

The randomized region is obtained by using a mixture of all four bases in each synthesis step. In most cases, the four building blocks are mixed with balanced stoichiometry (IUB mix code N ). However, pools may also be synthesized for the selection of aptamers that contain only three different bases in the randomized region [12] or unequal frequencies of the four bases [13]. 

To prevent the selection of aptamers with low affinity to the target molecule, the target concentration has to be decreased and the washing procedure has to be done with increased stringency. This enhances the discrimination between aptamers with high affinity and aptamers with lower affinity [6]. 

Partitioning matrix The choice of matrix for the immobilization of ligand is dependent on the tag on the ligand (see Note 1). In our case, for the selection of aptamers against proteins, we find that His-tagged recombinant proteins are the best choice. Thus, we used Ni-NTA beads (Qiagen, Canada) as our matrix. 

A number of methods for partitioning selected sequences from the background, which are chosen empirically by researchers, are available. For the selection of aptamers against proteins,... 

Only a few general points are addressed here. The reader is referred to a recent review by Gopinath (2007) for details on procednres for the selection of aptamers. Like any other combinatorial method, the SEFEX methodology first reqnires synthesis of the library. Compared to other libraries, it is easy to prepare an unbiased pool of DNA sequences, as the conpling efficiency of the A, T, G, or C phosphoramidite is very similar. One conld compensate for the slightly different incorporation of nucleotides a mixture of phosphoramidites in a ratio of... 

Berezovski, M. V., Mnsheev, M. U., Drabovich, A. P., Jitkova, J. V., Krylov, S. N. (2006b). Non-SELEX selection of aptamers withont intermediate amphfication of candidate ohgonncleotides. Aaf Protoc 1, 1359-1369. 

Automated selection of aptamers against protein targets translated in vitro from gene to aptamer. Nucleic Acids Res 30, el08. 

Kawakami, J., Imanaka, H., Yokota, Y., Sugimoto, N. (2000). In vitro selection of aptamers that act with Zn2-E. J Inorg Biochem 82, 197-206. 

Mosing, R. K., Mendonsa, S. D., Bowser, M. T. (2005). Capillary electrophoresis-SELEX selection of aptamers with affinity for HIV-1 reverse transcriptase. Anal Chem 77, 6107-6112. 

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