%0 Journal Article
%T Real-Time PCR-Coupled CE-SELEX for DNA Aptamer Selection
%A Patrick Ruff
%A Rekha B. Pai
%A Francesca Storici
%J ISRN Molecular Biology
%D 2012
%R 10.5402/2012/939083
%X Aptamers are short nucleic acid or peptide sequences capable of binding to a target molecule with high specificity and affinity. Also known as ¡°artificial antibodies,¡± aptamers provide many advantages over antibodies. One of the major hurdles to aptamer isolation is the initial time and effort needed for selection. The systematic evolution of ligands by exponential enrichment (SELEX) is the traditional procedure for generating aptamers, but this process is lengthy and requires a large quantity of target and starting aptamer library. A relatively new procedure for generating aptamers using capillary electrophoresis (CE), known as CE-SELEX, is faster and more efficient than SELEX but requires laser-induced fluorescence (LIF) to detect the aptamer-target complexes. Here, we implemented an alternative system without LIF using real-time- (RT-) PCR to indirectly measure aptamer-target complexes. In three rounds of selection, as opposed to ten or more rounds common in SELEX protocols, a specific aptamer for bovine serum albumin (BSA) was obtained. The specificity of the aptamer to BSA was confirmed by electrophoretic mobility shift assays (EMSAs), an unlabeled competitor assay, and by a supershift assay. The system used here provides a cost effective and a highly efficient means of generating aptamers. 1. Introduction Aptamers are short single-stranded oligomers made up of DNA, RNA, or peptides that are capable of binding a target ligand (proteins, small molecules, or even living cells) with high affinity. They are also known as artificial antibodies because in addition to binding with high affinity, they also bind with high specificity. Aptamers have several advantages over antibodies, including ease and low cost of production which does not involve animals. Aptamers are less immunogenic than antibodies and are already being used as therapeutic agents in humans [1]. Nucleic acid aptamers are also able to act in ways that antibodies cannot. Nucleic acid aptamers, unlike antibodies, can be selected for and used under nonphysiological conditions, such as high-salt conditions and varying pH [2]. Also, nucleic acid aptamers are able to undergo specific conformational changes that antibodies cannot. For example, nucleic acid aptamer binding can be ¡°turned off¡± by the addition of the complementary strand [3]. Additionally, nucleic acid aptamers can undergo a conformational change when binding to their target and can be used as molecular beacons, fluorescently ¡°off¡± when unbound and ¡°on¡± when bound [4]. The field of aptamers is rapidly growing as is the number of
%U http://www.hindawi.com/journals/isrn.molecular.biology/2012/939083/