Selection Process

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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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.

X-Aptamer Selection Process Diagram

FAQ: Selection Process

How does the X-Aptamer Selection Kit work?
The most important thing to know about the X-Aptamer Selection Kit is that it is not based on SELEX. AM Biotech has developed a two-step, bead-based method to quickly and easily identify sequences in the library that bind well with the target(s). The key to the X-Aptamer Selection Kit is the library of billions of microbeads. Each microbead arries about 0.1 picomole of just one unique, chemically-modified oligonucleotide. One or more of those chemically-modified oligonucleotides may be an X-Aptamer for a target and the kit protocol makes finding that bead/sequence relatively easy.
What do I need to perform a selection with a kit?
The X-Aptamer selection process uses simple tools and reagents typically found in most biochemistry laboratories. You will need access to items such as pipettes, buffers, a simple magnetic stand, a thermocycler, gel electrophoresis equipment, and a centrifuge.
What kinds of targets are compatible with the kit?
Currently, the X-Aptamer Selection Kit is designed to work with proteins, structurally stable peptides, and small molecules.
How long does it take to complete the kit protocol?
A scientist can complete the protocol in two to four days depending upon his/her skill level and familiarity with some of the protocol steps.
What skills/training do I need to complete the kit protocol?
The kit protocol can be successfully completed by a scientist or lab technician who has some training and/or experience with basic biochemistry laboratory techniques. No special training is required. Undergraduate students have successfully completed the kit with some guidance from an instructor.
What is the success rate?
For one selection using one library, the success rate is about 60%. This compares favorably with SELEX, which has a published success rate of between 30% and 50%. Additional selection(s) using different conditions and/or a different library may be successful.
How many X-Aptamers will I get from a kit?
Typically, for each target you used in the kit selection AM Biotech will send around five putative X-Aptamers. If you use the kit to select X-Aptamers to five targets simultaneously, then you will receive about 5 putative X-Aptamers per target (up to 25 total). If you use the kit to select X-Aptamers to just one target, you may receive 5 to 10 putative X-Aptamers to that single target. You will test these putative X-Aptamers for binding to your target(s). Some of the putative X-Aptamers that you receive may not bind to your target (false positives).
Will I get the sequences for the X-Aptamers?
This depends on which purchase option you choose. For the Co-Development Option in which AM Biotech provides sample processing for no charge, AM Biotech will send the sequence information to you after you send AM Biotech the identity of the targets used with the kit and the data that conclusively confirms binding to the targets. For the Standard Pricing Option, AM Biotech will provide the sequence information upon purchase of a commercial use license. A customer can use the X-Aptamers provided for unlimited research without a commercial license. Universities and other non-profit organizations can receive the sequence and modification information if required for acceptance of a publication in a scientific journal. However, if the university or non-profit does not submit a patent application for the X-Aptamer sequences, then AM Biotech will ask for performance data in return as well as the non-exclusive right to commercialize the X-Aptamers.
Can I get additional X-Aptamer material without a commercial license?
Yes. AM Biotech will synthesize additional X-Aptamer material for you at a reasonable price that would be comparable to what other oligo synthesis companies would charge for a similar oligonucleotide.