AM Biotech uses a proprietary bead-based technology to select X-Aptamers. The single round, bead-based discovery process has significant advantages relative to Systematic Evolution of Ligands by Exponential Enrichment (SELEX) approaches. AM Biotech’s process eliminates the repeated rounds of PCR amplification that are required with SELEX. This eliminates problems associated with PCR bias, which disfavors recovery of the most structurally stable aptamers. Significantly, the bead-based process is compatible with any type of DNA or RNA modification that can be incorporated by chemical synthesis. This includes a wide range of modifications that cannot be incorporated during the PCR steps in SELEX such as the phosphorodithioate backbone modifications. AM’s process also allows selection of X-Aptamers that contain more than one type of modification per DNA base. For example, one oligonucleotide can contain a normal T, a tryptophan-modified T, as well as other modified versions of T in any combination within the sequence. Such modified X-Aptamers are not compatible with PCR because with PCR, every T must either be normal DNA or the same modified version.
In the bead-based process, conventional solid-phase oligonucleotide synthesis combined with split-and-pool methods create combinatorial libraries in which each bead carries about 1012 copies of a single sequence, while the sequence varies between different beads. The oligonucleotides are covalently attached to the beads but through a special nucleotide at the 3’ end that enables detaching the oligos at a later stage. Primer sequences are included at each end to allow the recovery of the unmodified version of the sequence from selected beads.
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Watch our animation on the selection process.
Specific binding agents (X-Aptamers) are recovered by incubating a bead library with 1 to 5 targets of interest and recovering individual beads that preferentially bind the target(s). The oligos are detached from the selected beads, split into fractions, and each target is used individually to pull down the binding sequences from solution. The sequences that bind are converted to natural DNA, sequenced and analyzed. Sequences that are enriched for each target are synthesized with the specific chemical modifications that were used in the starting library and characterized for binding and specificity.
Much of the selection process can be readily performed by our customers, enabling the X-Aptamer Selection Kit.