Cambio - Excellence in Molecular Biology

Aptamers

Aptamers

Aptamers are single-stranded DNA or RNA molecules that can bind to pre-selected targets including proteins and peptides with high affinity and specificity. We offer catalogue aptamers (below); see the description tab (below) for details of our custom aptamer service.

Aptamers are single-stranded DNA or RNA (ssDNA or ssRNA) molecules that can bind to pre-selected targets including proteins and peptides with high affinity and specificity.  These molecules can assume a variety of shapes due to their propensity to form helices and single-stranded loops, explaining their versatility in binding to diverse targets.  They are used as sensors, and therapeutic tools, and to regulate cellular processes, as well as to guide drugs to their specific cellular targets.  Contrary to the actual genetic material, their specificity and characteristics are not directly determined by their primary sequence, but instead by their tertiary structure.

We supply aptamers against small molecules (eg antibiotics), proteins involved in the immune system, as well as a range of other targets.

Custom Aptamer Service

In addition to our presynthesised aptamer catalogue we now offer a service for the synthesis of custom high affinity aptamers against your favourite target through Base Pair Biotechnologies.  Our service takes advantage of a unique form of multiplex SELEX technology and we can make RNA or DNA apatamers depending on your needs.  Surprisingly, Base Pair Biotechnologies and others have found that in addition to RNA aptamers, DNA aptamers can also be generated with high target affinities, the latter allowing for greater stability.  Aptamers offer significant advantages over antibodies for many applications.

 

Enabled by a proprietary high-throughput aptamer selection process, we offer a flexible pricing structure for DNA aptamer development depending on your number of custom targets (recombinant proteins or synthetic peptides).  For example, if you have 16 targets, your price can be as little as $5000 per custom aptamer.

All prices include quantitative binding validation (Kd determination) and access to monoclonal aptamer material to validate in your assay.  Additional aptamer material can be purchased for approximately $200-500/100μg.  Other modifications including biotinylation, fluorophores, and other functionalizations are also available.

Intellectual Property transfer agreements are available for “no strings attached” control of full “monoclonal” aptamer sequence information.

Aptamers are typically selected against a single immobilized target during a process that can take weeks to months.

For further information please email or phone technical support at Cambio (Tel: +44 (0)1954 210200) and see the links below:

Advantages of aptamers over antibodies

Aptamers against small molecules

Frequently Asked Questions (FAQs)

 

Structure of aptamer developed by Base Pair Biotechnologies to IL7

 

Structure of IL-7 aptamer
ACKNOWLEDGEMENTS:
The following programs were utilized to generate the above structure:
[1] T. Macke and D.A. Case. Modeling unusual nucleic acid structures. In Molecular Modeling of Nucleic Acids, N.B. Leontes and J. SantaLucia, Jr., eds. (Washington, DC: American Chemical Society, 1998), pp. 379-393.
[2]. Visual Molecular Dynamics. 

 

Advantages of aptamers over antibodies

While aptamers are analogous to antibodies in their range of target recognition and variety of applications, they possess several key advantages over their protein counterparts [1]:

  • They are self-refolding, single-chain, and redox-insensitive.  They also lack the large hydrophobic cores of proteins and thus do not aggregate.  They tolerate (or recover from) pH and temperatures that proteins do not.
  • They are easier and more economical to produce (especially at the affinity reagent scale).  In stark contrast to peptides, proteins and to some small chemicals, oligonucleotides ( = DNA aptamers) are made through chemical synthesis, a process that is well defined, highly reproducible, sequence independent and can be readily and predictably scaled up.  Their production does not depend on bacteria, cell cultures or animals.
  • Related to the above, aptamers are much easier and less expensive to provide in GMP grade. This may be highly significant for applications such as cell sorting (FACS or MACS) in which agents come in contact with a potentially therapeutic cell line.
  • In contrast to antibodies, toxicity and low immunogenicity of particular antigens do not interfere with the aptamer selection.  Further, highly custom or “orphaned” targets can be addressed rapidly and cheaply.
  • In contrast to in vivo antibody selection, ON- and OFF-rates can be optimized for such varied applications as lateral flow assays and (aptamer) ELISAs.
  • They are capable of greater specificity and affinity than antibodies [2].
  • They can easily be modified chemically to yield improved, custom tailored properties.  For instance, reporter and functional groups and PEG can easily be attached to the aptamer in a deterministic way.  In fact, they can even be combined with antibodies [3, 4].  Similarly, their ADME properties can be readily tuned by conjugation to other groups (PEG, etc).
  • Their small size leads to a high number of moles of target bound per gram, and they may have improved transport properties allowing cell specific targeting and improved tissue penetration [5-9].
  • They are much more stable at ambient temperature than antibodies yielding a much higher shelf life, and they can tolerate transportation without any special requirements for cooling, eliminating the need for a continuous cold chain.

 

References:

1. Stoltenburg R, Reinemann C, Strehlitz B: SELEX–a (r)evolutionary method to generate high-affinity nucleic acid ligands. Biomolecular engineering 2007, 24:381-403.

2. Jayasena SD: Aptamers: An Emerging Class of Molecules That Rival Antibodies in Diagnostics. Clinical Chemistry 1999, 45:1628-1650.

3. Ferreira CS, Papamichael K, Guilbault G, Schwarzacher T, Gariepy J, Missailidis S: DNA aptamers against the MUC1 tumour marker: design of aptamer-antibody sandwich ELISA for the early diagnosis of epithelial tumours. Analytical and bioanalytical chemistry 2008, 390:1039-50.

4. Burbulis I, Yamaguchi K, Yu R, Resnekov O, Brent R: Quantifying small numbers of antibodies with a “near-universal” protein-DNA chimera. Nature Methods 2007, 4:1011-3.

5. McCauley TG, Hamaguchi N, Stanton M: Aptamer-based biosensor arrays for detection and quantification of biological macromolecules. Analytical biochemistry 2003, 319:244-50.

6. Cao Z, Tong R, Mishra A, Xu W, Wong GCL, Cheng J, Lu Y: Reversible Cell-Specific Drug Delivery with Aptamer-Functionalized Liposomes. Angew. Chem. Int. Ed. 2009, 48:6494-6498.

7. De Rosa G, La Rotonda MI: Nano and Microtechnologies for the Delivery of Oligonucleotides with Gene Silencing Properties. Molecules 2009, 14:2801-2823.

8. Ferreira CSM, Cheung MC, Missailidis S, Bisland S, Gariepy J: Phototoxic aptamers selectively enter and kill epithelial cancer cells. Nucl. Acids Res. 2009, 37:866-876.

9. Yan AC, Levy M: Aptamers and aptamer targeted delivery. rnabiology 2009, 6:316-320.

 

Aptamers against small molecules 

BasePair biotechnologies also have significant experience in the development of aptamers to non-protein targets including peptides and small molecules. A key component of such aptamer selection is proper immobilization and presentation of the target. BasePair Bio have the necessary chemistry and bioconjugation expertise to tackle the most challenging projects and can work with you on a case-by-case basis to develop your aptamer. Finally, BasePair Bio have significant biosensor/assay development experience and can recommend strategies for sensor development using your new aptamer(s).

  

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  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. 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 serumNucleic 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 MidkineJournal 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 IXaNature 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 vivoNat. 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.



For further information please call Cambio Technical Support  (01954 210200) or send us a message via our contact us page.