Semi-automated selection of DNA aptamers using magnetic particle handling

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Tony Rook
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Semi-automated selection of DNA aptamers using magnetic particle handling

Below is a reference to a recent protocol published in Biotechniques. The authors describe a robot assisted semi-automated method for selecting and isolating DNA aptamers.

Aniela Wochner, Birgit Cech, Marcus Menger, Volker A. Erdmann, and Jrn Glkler
Semi-automated selection of DNA aptamers using magnetic particle handling. BioTechniques September 2007
Volume 43, Number 3: Advanced Online Publication

Abstract
We have developed a semi-automatic selection procedure for DNA aptamers. Employing a robotic workstation for magnetic particle handling, this method allows for a fast, reproducible, and parallelized selection of DNA aptamers. The selection protocol is designed to provide high flexibility and versatility in terms of choice of buffers and reagents, as well as stringency of selection. Using this procedure, we have successfully isolated ligand-specific, high-affinity DNA aptamers.

INTRODUCTION
Aptamers are single-stranded DNA or RNA molecules that specifically bind to a certain target molecule with high affinity and specificity (1,2). Their application is very diverse: aptamers have been used as molecular recognition elements in analytical systems for detection, separation, or purification of target molecules (39). They also play an important role in medical therapy (10) and environmental analysis (11). Aptamers are usually isolated from a combinatorial library of nucleic acids by an in vitro selection method called systematic evolution of ligands by exponential enrichment (SELEX).

However, this procedure requires repetitive cycles and is therefore very time-consuming. For RNA aptamers, the selection process has been facilitated and accelerated by Cox and co-workers, who established an automated selection procedure (1214) based on magnetic beads. This process has been adjusted and optimized by several others (1316). An automated selection procedure for RNA (17) and DNA (18) aptamers without magnetic beads has also been reported.

Due to its 2′-hydroxyl group, however, RNA is easily subjected to hydrolysis by RNases, which can diminish its applicability if it is not stabilized by chemical modifications (1925). An alternative is the employment of Spiegelmers (L-RNAs), which represent the mirror images of the naturally occurring D-RNAs. Since the L-forms of nucleic acids do not occur in nature, there are no enzymes to degrade them (26,27). DNA aptamers, lacking the 2′-hydroxyl group, are also more stable than unmodified RNAs in biological samples.

Here, we describe a robot-assisted, semi-automated selection procedure for DNA-aptamers. This procedure exploits the advantages of automation (i.e., it allows for a fast, reproducible, and parallelized selection of DNA aptamers). Only little manual handling is required. Merely the preparation of the solutions, precipitation, and amplification steps have to be performed manually. All other selection steps, like coupling of the target to the solid surface, interaction of DNA with the target molecule, wash and elution steps, as well as the single-stranded DNA preparation can be carried out automatically. We have used this selection procedure to isolate specifically binding DNA aptamers.