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J Vis Exp. 2010 Jul 26;(41). pii: 2039. doi: 10.3791/2039.

Primer-free aptamer selection using a random DNA library.

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  • 1Department of Pathology, Hershey Medical Center, Pennsylvania State University, PA, USA.

Abstract

Aptamers are highly structured oligonucleotides (DNA or RNA) that can bind to targets with affinities comparable to antibodies (1). They are identified through an in vitro selection process called Systematic Evolution of Ligands by EXponential enrichment (SELEX) to recognize a wide variety of targets, from small molecules to proteins and other macromolecules (2-4). Aptamers have properties that are well suited for in vivo diagnostic and/or therapeutic applications: Besides good specificity and affinity, they are easily synthesized, survive more rigorous processing conditions, they are poorly immunogenic, and their relatively small size can result in facile penetration of tissues. Aptamers that are identified through the standard SELEX process usually comprise approximately 80 nucleotides (nt), since they are typically selected from nucleic acid libraries with approximately 40 nt long randomized regions plus fixed primer sites of approximately 20 nt on each side. The fixed primer sequences thus can comprise nearly approximately 50% of the library sequences, and therefore may positively or negatively compromise identification of aptamers in the selection process (3), although bioinformatics approaches suggest that the fixed sequences do not contribute significantly to aptamer structure after selection (5). To address these potential problems, primer sequences have been blocked by complementary oligonucleotides or switched to different sequences midway during the rounds of SELEX (6), or they have been trimmed to 6-9 nt (7, 8). Wen and Gray (9) designed a primer-free genomic SELEX method, in which the primer sequences were completely removed from the library before selection and were then regenerated to allow amplification of the selected genomic fragments. However, to employ the technique, a unique genomic library has to be constructed, which possesses limited diversity, and regeneration after rounds of selection relies on a linear reamplification step. Alternatively, efforts to circumvent problems caused by fixed primer sequences using high efficiency partitioning are met with problems regarding PCR amplification (10). We have developed a primer-free (PF) selection method that significantly simplifies SELEX procedures and effectively eliminates primer-interference problems (11, 12). The protocols work in a straightforward manner. The central random region of the library is purified without extraneous flanking sequences and is bound to a suitable target (for example to a purified protein or complex mixtures such as cell lines). Then the bound sequences are obtained, reunited with flanking sequences, and re-amplified to generate selected sub-libraries. As an example, here we selected aptamers to S100B, a protein marker for melanoma. Binding assays showed Kd s in the 10(-7) - 10(-8) M range after a few rounds of selection, and we demonstrate that the aptamers function effectively in a sandwich binding format.

PMID:
20689511
[PubMed - indexed for MEDLINE]
PMCID:
PMC3156072
Free PMC Article
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