Aptamers and the next generation of diagnostic reagents

Abstract

Antibodies have been extensively used as capture and detection reagents in diagnostic applications of proteomics-based technologies. Proteomic assays need high sensitivity and specificity, a wide dynamic range for detection, and accurate, reproducible quantification with small confidence values. However, several inherent limitations of monoclonal antibodies in meeting the emerging challenges of proteomics led to the development of a new class of oligonucleotide-based reagents. Natural and derivatized nucleic acid aptamers are emerging as promising alternatives to monoclonal antibodies. Aptamers can be effectively used to simultaneously detect thousands of proteins in multiplex discovery platforms, where antibodies often fail due to cross-reactivity problems. Through chemical modification, vast range of additional functional groups can be added at any desired position in the oligonucleotide sequence, therefore the best features of small molecule drugs, proteins, and antibodies can be brought together into aptamers, making aptamers the most versatile reagent in proteomics. In this review, we discuss the recent developments in aptamer technology, including new selection methods and the aptamers' application in proteomics.

Figure 1

Chemical structures of modified oligonucleotides. (A) normal, phosphodiester backbone (B). mono-thio modified thioaptamer (C) di-thio modified thioaptamers (D) di-thio modified X-aptamer.

Figure 2

Scheme showing the traditional SELEX procedure. The iterations are continued until the sequences converge and the tightly binding sequences are selected and identified. For the RNA oligonucleotides, two additional steps are needed for each iteration. The selected RNA sequences are converted and amplified into DNA sequences via RT-PCR, and then converted back into RNA sequences via in vitro transcription.

Figure 3

Scheme showing the bead-based selection process. In the first step, the bead based library is incubated with fluorescently tagged protein. In the second step, the protein bound beads are separated. Third step shows the protein is stripped from the bead. The sequence on the bead is then PCR-amplified, cloned and sequenced. The bottom panel shows the bead-based aptamer library screen. (A) An aliquot of bead-based library is viewed under light microscope. (B). The same filed viewed under fluorescent microscope. The aptamer bead bound to the protein is easily identified among the many hundreds of non-reactive beads. (C). A single positive bead is retrieved using a hand-held micropipette under fluorescent microscope.