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Frequently Asked Questions (FAQs) | stg.bpb.bpb.cgstack.com

Frequently Asked Questions (FAQs)

1. On Base Pair’s Success Rate: "If we invest in Base Pair to develop an aptamer, it would be important to know the success rate or your track record. It would be very helpful if you can give us a few examples (such as published papers) where your aptamers are used by 3rd party research groups."

Answer: Since beginning our commercial aptamer discovery services, we have had successes with a number of customers. Prior to this, we have developed numerous functional aptamers on government grants and contracts. Much of the private work we have performed is protected under non-disclosure agreements, however, we can point to a considerable amount of publications and federal funding for our aptamer development and characterization. One of our current “customers” is the National Cancer Institute, and we expect considerable additional publishable data to soon result from this and other efforts. In addition we have shared in our catalogs validated aptamers that have not been sequestered by their requestors for their exclusive use.

2. Low discovery pricing: "How can you offer such low discovery pricing compared to other aptamer companies?"

Answer: Traditionally SELEX has been done with a single target and single library. Base Pair has developed and proven a patent pending, multiplex target approach that allows us to execute selections more cost effectively. We also have a progressive ownership model that allows for increasing investment based on amount of aptamer control desired.

3. Project progress: "How will I know the status of my project and what if I have questions?"

Answer: Base Pair has a proprietary project management portal the AptaTracker, where for each project researchers are invited to join a confidential chat environment to interact with Base Pair scientists which includes weekly updates and data sharing.

4. On Aptamers to test: "What does Base Pair provide? How many clones will we get to evaluate?"

Answer: Targets such as proteins, peptides, small molecules, and cells of interest are provided by researchers as targets for our SELEX process for a low discovery price. Once aptamers are discovered and validated by SPR/BLI/MST or other method they are available to the researcher for evaluation in their assay. If successful, aptamer material can either be purchased from us, the sequence can be transferred for publication or research purposes, or the intellectual property can be transferred outright for commercialization purposes. While our process typically yields multiple clones per target as valid aptamer candidates, for our low discovery prices, we can only provide Kd determination on a few clonal aptamers validated to bind. We can, however, provide these and additional clones to the customer for additional screening at a nominal cost (~$300/100 microgram).

5. Can you provide aptamer sandwich pairs?

Answer: Depending on the target sandwich pairs (multiple aptamers that bind the same target at different locations) can be screened for and made available with a variety of modifications for testing.

6. On aptamer performance/expected Kd: "What affinity can we expect for our target? Based on the literature, it seems that low nM affinity is common for protein targets. Is that the performance we can expect?

Answer: Every target is different, but we offer a 50% return policy if we cannot verify an affinity by SPR or BLI or other method. Depending on the target, we often do much better (picomolar range). We have a publicaly available list of aptamers in our catalogs with a range of binding constants. These lists reflect aptamers developed under a variety of projects over a considerable period of time and is intended to represent a range of target classes. Several aptamer clones (sequences) may need to be validated for very small or very large targets (such as small molecules or very large proteins, respectively) to identify an aptamer with the best Kd. We have the high-throughput instrumentation necessary to perform such validation. Most importantly, we have implemented additional quality control in which apparent affinities are measured at the polyclonal stage of the process. Through this feedback during the selection process, we can verify that enrichment of “good” binders is occurring for your target. Finally, we should emphasize that a small Kd may not be the only determinant for success depending on your application, especially in the case of competitive binding or reversible sensing applications.

7. On Difficult Targets: "Can you select aptamers to small molecules"

Answer: Base Pair has been able to generate aptamers successfully to several small molecules (see Available Catalog Aptamers) and several of our current customers have contracts for development to others. Perhaps the most important aspect of selecting aptamers is target presentation. Base Pair has the expertise and collaborators to work with our clients to identify the best small molecules and small molecule derivatives for target presentation, bioconjugation, and even sensor development.

“Are peptides viable targets for aptamer selection?”

Answer: This is a valid point and one that has been sited when peptides fail as successful antigens for antibody development. As such, a similar risk exists when using a peptide as an “antigen” for aptamer development. One would presume that the longer the peptide is, and the better fidelity a peptide has to its context in the larger protein, the greater the probability of success. There is actually a large body of bioinformatic work and programs for selecting peptides as subsequences/surrogates for whole proteins. Given these issues, we generally rely on our customers to select peptides as targets. While we cannot guarantee aptamer binding in the context of the larger protein, we generally can arrive at tightly binding aptamers to the peptides themselves and provide multiple clonal candidates for testing purposes.

8. On DNA aptamer stability in vivo: "We’re planning an in vivo (or cell culture) application … will my DNA aptamer remain stable?"

Answer: The use of aptamers in vivo or in cell culture is generally challenged by the susceptibility of unmodified nucleic acids to degradation by nucleases. In particular 3’-exonuclease activity has been found to be the most prevalent nuclease activity both in calf and human serum [1]. Takei et al. [2] have shown that the degradation of unmodified DNA oligonucleotides in serum begins within an hour after administration, and that the oligonucleotide is completely removed within 24 h. However, when the same oligo was synthesized with a 3’-inverted thymidine (readily available from several synthesis companies) the degradation in the blood stream is significantly delayed (to approx. 72 hours) [2]. Thus, for many in vivo or cell culture applications, simple 3’-terminal modifications are likely to confer acceptable resistance to nuclease degradation.

Of course, immobilization of an aptamer (on beads for example) is likely to further extend serum half-life, and the free-solution end of the aptamer can be readily modified as well (often by pegylation, for instance).

Unlike RNA aptamers which must routinely be modified at the 2’- position to protect from RNA endonucleases [3-5], end-protected DNA aptamers are likely to remain stable for long time periods [2].

9. To test the technology: "Do you have existing validated binders for evaluation?"

Answer: We have been able to generate multiple target aptamers which are available for validation in various assays, some are available for commercial exploration as well. For minimal cost these aptamers can be purchased for testing, a list can be found here. We also have developed an the AptaColor(TM) Kit so our aptamers can be applied in a pre-defined lateral flow assay format.

10. On aptamer ownership: "Do we (the customer) gain access to the sequence of our aptamer? And if so, are there extra costs involved? Do we have exclusive rights to the aptamer or can you sell it to others? Can we produce the aptamer in our lab after you isolate it or do we buy from you as we need it?"

Answer: Ownership of aptamer sequences is negotiated on a case-by-case basis. In our experience, however, three general cases often apply:

Case #1 – "No sequence rights necessary": As with monoclonal antibodies, many researchers simply need a high affinity ligand to a target of interest. Researchers in this case rarely know (or even care to know) the amino acid sequence of their antibody. We can provide aptamer material as needed. Additionally, many researchers are becoming increasingly aware of the potential advantages of aptamers over antibodies. Our fast turnaround time for aptamer production (4-6 weeks) vs. production of a custom monoclonal antibody (3-6 months) can be a major motivating factor as well.

Case #1b – "… but I need to publish the aptamer sequence for my research paper …": We understand and want to help! Many aptamers have been used to gain a better understanding of nucleic acid and other molecular recognition events. For such research, knowledge of the aptamer sequence can be crucial. In these cases, access to the sequence can be given. Base Pair retains all intellectual property rights to the aptamer sequence and would request being acknowledged or cited in any paper.

Case #2 – "We’re developing a new diagnostic or therapeutic … we’re not sure where this could lead, but if it works, we will need an exclusive license to the sequence": Let Base Pair supply your research materials and provide the sequence when it really becomes necessary. In this case we supply a relatively simple Material Development Agreement (MDA) in which you agree to not reverse engineer our aptamer materials during evaluation. Base Pair retains intellectual property rights to the aptamer sequence until YOU decide an exclusive license or buyout makes sense. There will be no surprises as you’ll already know what this transfer of rights will cost before we start. We understand flexibility is needed in such situations and are open to a variety of license and ownership agreements.

Case #3 – "BasePair’s aptamer is just one piece of a much larger puzzle. We can’t license anything – we need to own it outright." That’s great. We think you will still find that we can supply your necessary affinity ligand at a competitive overall price, even if you need to buy it "lock, stock, and barrel".

REFERENCES:
1. Shaw JP, Kent K, Bird J, Fishback J, Froehler B: Modified deoxyoligonucleotides stable to exonuclease degradation in serum. Nucleic acids research 1991, 19:747-50.
2. Takei Y, Kadomatsu K, Itoh H, Sato W, Nakazawa K, Kubota S, Muramatsu T: 5′-,3′-Inverted Thymidine-modified Antisense Oligodeoxynucleotide Targeting Midkine. Journal of Biological Chemistry 2002, 277:23800 -23806.
3. Rusconi CP, Scardino E, Layzer J, Pitoc GA, Ortel TL, Monroe D, Sullenger BA: RNA aptamers as reversible antagonists of coagulation factor IXa. Nature 2002, 419:90-94.
4. Rusconi CP, Roberts JD, Pitoc GA, Nimjee SM, White RR, Quick G, Scardino E, Fay WP, Sullenger BA: Antidote-mediated control of an anticoagulant aptamer in vivo. Nat. Biotechnol2004, 22:1423-1428.
5. Potti A, Rusconi CP, Sullenger BA, Ortel TL: Regulatable aptamers in medicine: focus on antithrombotic strategies.Expert opinion on biological therapy 2004, 4:1641-7.



Researcher

University of Parma, Italy

“Base Pair Biotechnologies provided us a tailored solution for our aptamer needs in a fast, efficient and professional manner."