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 (3,4,5,6,7,8,9). 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 (12,13,14) based on magnetic beads. This process has been adjusted and optimized by several others (13,14,15,16). 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 (19,20,21,22,23,24,25). 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.

Materials and Methods

Materials

A single-stranded DNA oligonucleotide library with a theoretical diversity of 10¹⁴ was used as precursor pool for the aptamer selection. The 40 nucleotide random sequence was flanked by 20 nucleotide primers on either side. 5′-GGGAATTCGAGCTCGGTACC-3′ was used as forward primer, 5′-biotin-C6-CCAAGCTTGCATGCCTGCAG-3′ as reverse primer. All oligonucleotides were obtained from Purimex (Grebenstein, Germany). GoTaq® Flexi DNA Polymerase (Promega GmbH, Mannheim, Germany) was used for PCR during selection procedure. Magnetic beads (Dynabeads® M-280 Streptavidin) were purchased from Invitrogen GmbH (Karlsruhe, Germany). Sulfo-NHS-SS-Biotin was supplied by Perbio Science Deutschland GmbH (Bonn, Germany), and Triton® X-100 was supplied by Arcos Organics (Geel, Belgium). Plasticwares for BioSprint 15 were purchased from Qiagen GmbH (Hilden, Germany). Biotin, daunomycin, and all other materials were obtained from Sigma-Aldrich Chemie GmbH (Munich, Germany).

Robotic Workstation

Aptamer selections were performed using the robotic workstation BioSprint 15 (Qiagen) (Figure 1). This automated device only transfers the isolated magnetic particles and not, like other workstations, the bead suspension, and it enables the parallel execution of up to 15 different selections. BioSprint 15 comprises three rows of five magnetic rods each that are joined on the top end and that can be moved as a whole both horizontally and vertically. On the middle level, plastic covers can be introduced, which enclose the magnetic rods once they dip into the reaction solution. A removable tray contains plug-in positions for a total of 15 plastic strips, each consisting of five reaction tubes (tubes A–E).

Figure 1. Inside view into robotic workstation BioSprint 15. (Click to enlarge)

Using the magnetic rods, magnetic beads can be mixed in solution at different speeds (5–150 mm/s) and transferred between the different tubes of a plastic strip. The operating mode of BioSprint 15 is shown in Figure 2; covered by a plastic cover, the magnet dips into the tube containing suspended magnetic particles. By slow up-and-down movements, the magnetic particles are collected and can be transferred into the next tube. Here, the magnetic rod is removed from the plastic cover, and the magnetic particles are suspended in the new solution. The programs directing the BioSprint's movements were written using BioSprint software 1.0 (Qiagen).