FluMag-SELEX as an advantageous method for DNA aptamer selection

Abstract

Aptamers are ssDNA or RNA oligonucleotides with very high affinity for their target. They bind to the target with high selectivity and specificity because of their specific three-dimensional shape. They are developed by the so-called Systematic Evolution of Ligands by Exponential Enrichment (SELEX) process. We have modified this method in two steps-use of fluorescent labels for DNA quantification and use of magnetic beads for target immobilization. Thus, radioactive labelling is avoided. Immobilization on magnetic beads enables easy handling, use of very small amounts of target for the aptamer selection, rapid and efficient separation of bound and unbound molecules, and stringent washing steps. We have called this modified SELEX technology FluMag-SELEX. With FluMag-SELEX we have provided a methodological background for our objective of being able to select DNA aptamers for targets with very different properties and size. These aptamers will be applied as new biosensor receptors. In this work selection of streptavidin-specific aptamers by FluMag-SELEX is described. The streptavidin-specific aptamers will be used to check the surface occupancy of streptavidin-coated magnetic beads with biotinylated molecules after immobilization procedures.

First report of the use of a saxitoxin–protein conjugate to develop a DNA aptamer to a small molecule toxin

“…These short ribonucleic acid (RNA) or single-stranded deoxyribonucleic acid (ssDNA) analytical recognition elements are discovered from a starting random library of 10 12 -10 15 oligonucleotide sequences via an iterative process known as systematic evolution of ligands by exponential enrichment (SELEX; Ellington and Szostak, 1990;Tuerk and Gold, 1990). Separation of target bound and unbound oligonucleotides has been performed using strategies such as affinity chromatography (Liu and Stormo, 2005), filtration (Schneider et al, 1993), SPR sensors (Misono and Kumar, 2005), and magnetic bead immobilization (Bruno and Kiel, 2002;Stoltenburg et al, 2005). Selected aptamers have been demonstrated for use in assays and platforms that have traditionally employed antibodies as analytical recognition elements.…”

Design and Characterization of DNA Aptamer for Breast Tumor Marker by an Advantageous Method

“…In the selection procedure magnetic beads were used as an immobilization matrix for target protein (Stoltenburg et al, 2005) (Figure 1). Selection procedure was started by amplification of ssDNA library.…”

“…Washed beads were diluted in 240µL Buffer E [5mM sodium phosphate buffer (pH 7.6) with 0.1% Tween20] to achieve the final bead concentration 20 mg/mL. Magnetic beads are a very useful matrix for immobilization of targets during aptamer selection and enable easy and efficient processing (Stoltenburg et al, 2005).…”

“…Another kind of separation method is filtration, for example, using nitrocellulose filters (Tombelli et al, 2005b). The use of magnetic beads offers another possibility for target immobilization (Lupold et al, 2002;Kikuchi et al, 2003;Murphy et al, 2003;Stoltenburg et al, 2005). This method requires only very small amounts of target and enables a very simple handling.…”

Probing and quantifying DNA–protein interactions with asymmetrical flow field-flow fractionation

“…MD simulations were performed on the Stampede supercomputer resource (Texas Advanced Computing Center) provided by XSEDE Science Gateways with the Amber 12 program package. [38,39] An AMBER 99 force field [40] and TIP3P water model [41] were applied. The counter ions of Na + , Mg 2+ , K + and Cl -were used in modeling at the same concentrations as in the AF4 carrier solution to mimic the actual solution environment for the ssDNAs.…”