Strategies for the discovery of therapeutic aptamers

Abstract

Importance of the field: Therapeutic aptamers are synthetic, structured oligonucleotides that bind to a very broad range of targets with high affinity and specificity. They are an emerging class of targeting ligand that show great promise for treating a number of diseases. A series of aptamers currently in various stages of clinical development highlights the potential of aptamers for therapeutic applications.

Areas covered in this review: This review covers in vitro selection of oligonucleotide ligands, called aptamers, from a combinatorial library using the Systematic Evolution of Ligands by Exponential Enrichment process as well as the other known strategies for finding aptamers against various targets.

What the reader will gain: Readers will gain an understanding of the highly useful strategies for successful aptamer discovery. They may also be able to combine two or more of the presented strategies for their aptamer discovery projects.

Take home message: Although many processes are available for discovering aptamers, it is not easy to discover an aptamer candidate that is ready to move toward pharmaceutical drug development. It is also apparent that there have been relatively few therapeutic advances and clinical trials undertaken due to the small number of companies that participate in aptamer development.

Keywords: SELEX; aptamers; in vitro selection; oligonucleotide phosphorodithioate; thioaptamer.

Figure 1. General scheme of the SELEX or in vitro selection procedure

Figure 2. A negative-SELEX selected thioaptamer sequence binds more selectively to RelA/RelA than to p50/50 homodimers of NF-κB

Lanes 1–3 show the initial thioaptamer library, either incubated with RelA/RelA (Lane 1), incubated with p50/p50 (Lane 2), or with no added protein. Lane 4 is a selected thioaptamer clone isolated from the initial library after 10 rounds of selection for binding to RelA/RelA and negative-SELEX against p50/p50. Lanes 5, 6, and 7 represent the thioaptamer with 800, 400, and 200 nM RelA/RelA, respectively. Lanes 8, 9, and 10 represent the thioaptamer with 800, 400, and 200 nM p50/50, respectively. For a complex target such as a cell, the negative SELEX procedure is often included. In one example, a library of ssDNAs that contained a 52-mer random sequence was incubated with the T-cell acute lymphoblastic leukemia cell line CCRF-CEM to allow binding to take place [36]. The cells were then washed, and the DNA sequences bound to the cell surface were eluted. The collected sequences were then allowed to associate with excess negative control cells (B-cell line from human Burkitt’s lymphoma, Romos), and only the DNA sequences remaining free in the supernatant were collected and amplified for the next-round selection. After multiple rounds of selection, this negative-SELEX process efficiently reduced the DNA sequences bound to the control cells, while those target-cell-specific aptamer candidates were enriched [36].

Figure 3. Toggle SELEX

Aptamers that bind both human and mouse proteins are selected by toggling the protein target between human and mouse during alternating rounds of selection.

Figure 4. A bead-based aptamer library screen

(a) An aliquot of aptamer beads bound to target protein labeled with the Alexa Fluor 488 dye viewed under light microscopy. (b) The same beads viewed under fluorescence microscopy, in which a positive green bead stained with Alexa Fluor 488 dye can be easily identified in a background of many hundreds of nonreactive beads. (c) A single positive bead can easily be retrieved with a hand-held micropipette under a fluorescence microscope. Scale bar, 100 µm.