X-Aptamers are the next step in the evolution of aptamer technology. They are synthetic affinity reagents that incorporate natural as well as chemically-modified DNA or RNA nucleotides. Common modifications include amino acid functional groups as well as small molecules in virtually any combination. For many years, aptamers were developed using one of several versions of the Systemic Evolution of Ligands by Exponential Enrichment (SELEX) process. Unfortunately, SELEX has inherent limitations. The most notable being limited chemical diversity. Chemical diversity strongly influences the interaction between an affinity reagent and its target and directly affects binding affinity and specificity. Because SELEX relies on enzymatic amplification of an oligonucleotide library, only those chemically modified nucleotides that are compatible with PCR amplification can be used. In addition, the number of distinct, modified nucleotides that can be incorporated into any one SELEX library is severely constrained. This is in stark contrast to the vast chemical diversity of antibodies.
X-Aptamer technology overcomes SELEX limitations and exponentially expands the chemical diversity available for target interaction. X-Aptamers are developed using a patented microbead-based selection process synthesized using combinatorial chemistry as well as a patented synthesizer. In contrast to SELEX, the microbead process does not rely on PCR amplification of the binding sequences and thus enables a rich diversity of chemical functionality that is necessary to achieve excellent specificity and affinity. Many chemical modifications as well as combinations of those modifications that are not compatible with SELEX are available for use with AM Biotech’s bead-based X-Aptamer technology.
Affinity Molecule Comparison
FAQ: X-Aptamer Technology
What is an X-Aptamer?
An X-Aptamer is an affinity agent (similar in function to an antibody) that is comprised of a single strand of DNA with one or more (usually several) non-DNA chemical modifications attached to one or more of the DNA nucleobases and/or backbone. The most common modifications used in an X-Aptamer library are amino acid functional groups such as indole (tryptophan), phenol (tyrosine), and amine (lysine). Other modifications are also used. The customer also has the opportunity to include a novel modification in a library (such as known small molecule drugs to the target).
How is an X-Aptamer different than an aptamer?
An X-Aptamer has much greater chemical diversity than an aptamer. X-Aptamers use combinations of chemically-modified DNA bases and backbones that typically cannot be amplified using PCR. This makes it impossible to select X-Aptamers using the typical enzymatic processes used to select aptamers (e.g. SELEX). The diversity of the chemical functional groups in X-Aptamers provides much better interaction with a target, which typically results in better binding affinity and specificity. The two amino acids found to be most prevalent at the interface of a protein-protein contact are the hydrophobic amino acids tryptophan and tyrosine – these modifications are typically included in selection libraries. They tend to help to increase affinity and result in slow-off kinetics.
Why should I use an X-Aptamer instead of an antibody?
Antibodies are powerful and widely-used affinity agents, but they have limitations that X-Aptamers overcome. X-Aptamers are ambient stable, have a long shelf life, can be developed in non-physiological conditions, and can be developed for targets that are difficult for antibodies (toxins, small molecules, targets that do not illicit an antibody response, etc.)
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 carries about 20 femtomole 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 much easier than in the SELEX protocol.
Don’t you need a library of 10 ^14 sequences to be successful?
The short answer is no. X-Aptamer technology proves that! A much smaller library of sequences works quite well as long as the chemical diversity of the groups that interact with a target is enhanced. A standard meme in the SELEX community is that you need extraordinary sequence diversity. However, in reality, the sequence diversity that is actually present in a library is far lower than advertised. In addition, a large portion of that library never makes it to later SELEX rounds due to PCR bias.
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 small 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 necessary.
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 will receive about 5 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 reputable scientific journal.
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 modified oligonucleotide.