The X-Aptamer Selection Kit for Cells (XASK-C) is a powerful new technology that enables simple, rapid (one round) isolation of X-Aptamers that bind to a cellular target. The XASK-C uses a unique ‘nanobead’ library wherein each nanobead has attached many copies of just one chemically modified oligonucleotide sequence. A nanobead library can consist of as many as 1012 individual members.
There are numerous ways to set up a selection to cells. In one common way, the nanobead library is first incubated with one or more types of control cells (not the target cell). The library nanobeads that bind to the control cells are recovered for later processing while those that do not bind are used for the target cell selection.
The nanobeads that did not bind to the control cells are incubated with the targeted cells. Nanobeads that carry a sequence that binds to one or more features on the targeted cell surface will attach to the target cells. The nanobeads that do not bind to the targeted cells are removed through thorough washing and discarded. Those that are bound to the targeted cells are recovered.
The oligonucleotides from the nanobeads recovered from the control cells as well as from the targeted cells are released into solution into separate pools. Each pool of these oligos is split into two fractions and individually re-incubated with fresh control and targeted cells. After thorough washing, the oligonucleotides bound to the targeted cells as well as the control cells are amplified into unmodified DNA using sequence-coded primers.
The customer sends the PCR product to AM Biotech for sequencing using next generation methods (e.g. Ion Torrent, Illumina, etc.) and data analysis. AM Biotech identifies the sequences in the targeted cell fraction that are highly enriched when compared to the control cells. Those enriched sequences are synthesized and sent to the customer for screening against the targeted cells.
The cost of developing an X-Aptamer using AM Biotech’s kit is up to 10X less of what a typical contract research company would charge to perform an aptamer selection. Performing a selection using multiple targets in parallel further lowers the selection cost per target. In addition, a customer can save weeks of effort by using the kit’s rapid bead-based selection process instead of SELEX.
Contact us for the prices of the X-Aptamer Selection Kit and Processing Services
IMPORTANT NOTE: XA libraries contain primary amines. If your final application requires labeling the XA with an amine, then AM Biotech will need to send you a selection library that does not include primary amines. Methylene Blue functionalization requires use of a primary amine label.
FAQ: X-Aptamer Technology
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).
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.
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.)
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.
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.
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.
Currently, the X-Aptamer Selection Kit is designed to work with proteins, structurally stable peptides, and small molecules.
Yes; however, there are factors that you should consider. If a peptide has significant native structure (secondary or tertiary) in solution, then it is likely a good target to use with the kit. If a peptide is quite short (<20 aa), has no intramolecular disulfides and is essentially a flexible, randomly oriented chain, then successful X-Aptamer selection is less likely. In addition, if a peptide has a significant number of hydrophobic amino acids, then the opportunity to select an X-Aptamer improves since our unique library contains numerous hydrophobic groups. Please contact AM Biotech or one of our partners if you are not sure if you should use the X-Aptamer Selection kit for a selection against your peptide.
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.
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.
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.
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).
This depends on which purchase option you choose.
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.
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.