Automated selection of aptamers against protein targets translated in vitro: from gene to aptamer

Abstract

Reagents for proteome research must of necessity be generated by high throughput methods. Aptamers are potentially useful as reagents to identify and quantitate individual proteins, yet are currently produced for the most part by manual selection procedures. We have developed automated selection methods, but must still individually purify protein targets. Therefore, we have attempted to select aptamers against protein targets generated by in vitro transcription and translation of individual genes. In order to specifically immobilize the protein targets for selection, they are also biotinylated in vitro. As a proof of this method, we have selected aptamers against translated human U1A, a component of the nuclear spliceosome. Selected sequences demonstrated exquisite mimicry of natural binding sequences and structures. These results not only reveal a potential path to the high throughput generation of aptamers, but also yield insights into the incredible specificity of the U1A protein for its natural RNA ligands.

Combining SELEX and the yeast three-hybrid system for in vivo selection and classification of RNA aptamers

“…Since both techniques can be automated (Cox et al 2002;Eulberg et al 2005;Stelzl et al 2005), high-throughput applications also are feasible.…”

Aptamer-Based Electrochemical Sensing of Lysozyme

“…The first publications dealing with anti-lysozyme aptamers, selected by systematic evolution of ligands by exponential enrichment (SELEX) are from Ellington and co-workers [68][69][70] (Table 1). Based on these results, Kirby et al [70] have developed a reusable bead-based electronic tongue sensor arrays of anti-lysozyme aptamers for the detection of proteins, where fluorescence labeling is involved.…”

“…The main characteristics of the aptamers used in lysozyme biosensors are summarized in Table 1, starting from the first examples proposed by Ellington and co-workers [68,69], followed by Tran et al [71]. By using electrochemical impedance spectroscopy (EIS) on screen-printed carbon electrodes [63], it was demonstrated that the lowest detection limit and wider linear range can be achieved using the aptamer proposed by Tran et al [71].…”

Micromagnetic selection of aptamers in microfluidic channels

“…On the other hand, magnetic bead-based selection methods have been shown to be effective for virtually any class of molecular targets that can be immobilized on bead surfaces, including small molecules (26), proteins (27,28), and cell surfaces (21,29). However, the efficiency of magnetic selection techniques has lagged far behind what can be achieved by CE and other advanced molecular separation methods, and most current methods require multiple time-consuming selection rounds (27)(28)(29)(30)(31)(32)(33) or delicate manual manipulation of micron-sized magnetic beads (34).…”