Answers by Dr. Gregory Penner

Should I use an RNA or DNA aptamer?

I am not convinced that a DNA aptamer does not work as well as an RNA aptamer. The theory is that RNA aptamers are more flexible, and there has been considerable work done with RNA sequences to model tertiary structures. I think we are just beginning to scratch the surface regarding what tertiary structures single stranded DNA molecules are capable of forming. G-quartet structures make excellent aptamers with binding affinities similar to RNA aptamers.

For diagnostic applications an RNA aptamer will cost you more to synthesize commercially and will have less shelf life than an antibody based product. A natural nucleotide DNA aptamer will have a shelf life of a year at room temperature.

For therapeutic applications, it is necessary to use modified nucleotides to incorporate nuclease resistance. The enzymes used to process RNA (RNA polymerases and reverse transcriptase) work much better with these modified nucleotides than DNA polymerase works with modified DNA nucleotides. For this reason, we use modified RNA for therapeutic applications.

Should I consider using modified nucleotides?

For diagnostic applications I have yet to see data that demonstrates that the use of modified nucleotides leads to lower binding affinity than is possible with natural nucleotides. Our use of next generation sequencing in the discovery process enables us to identify the best natural nucleotide sequences available.

If you are serious about developing a commercial diagnostic platform with the aptamer you develop, you need to consider cost of production. With natural nucleotides you will have a lower cost of synthesis and you will have the freedom to have your aptamer synthesized by anyone without a royalty. For therapeutic applications you need to use modified nucleotides at all positions in the selection process and this will result in lower synthesis costs on your final commercial aptamer than with a mixture of nucleotides and will provide the level of nuclease protection necessary. You will also need to add mass to the aptamer to avoid it being flushed through the body too quickly.

Why are there not more aptamer based products commercialized?

There are several fairly simple reasons:

  • There were broad patents on the use of oligonucleotides that bound to a target molecule globally until 2011/12. The window of commercial opportunity has not been open for very long.
  • The development of commercial products from antibodies took decades to develop with several research groups working collaboratively. This has not been the case with aptamers.
  • There is a lack of awareness that aptamers cannot be plugged into antibody systems and expected to work. It is very easy to render an aptamers ineffective by immobilizing them to charged surfaces, or to proteins. We worked for years to develop methods to keep aptamers functional when immobilized.
  • All antibody based therapeutics are based on the stimulation of the immune response. This will not happen with aptamers, thus more clever approaches are necessary for aptamer based therapeutics.

Is it true that NeoVentures does not take an Intellectual Property position on aptamers that are developed for clients?

Yes, we think our competition is with antibody providers. Antibody providers do not take an IP position on antibodies that are custom developed for you. We do however invent the aptamers that we provide you with. As such, it is necessary that we be listed as inventors on patents that you file regarding these aptamers. As part of our supplier service agreement we provide clients with a royalty free, exclusive, global, irrevocable license to the aptamers that we provide them with for the application intended.

What is our success rate with aptamer development?

This is not a fair question as we cheat on the answer. We structure projects to share risk with clients. Part of your payment structure is based on our delivery of aptamers that meet agreed upon specifications. As such, we will not attempt to develop aptamers where we think that the risk of failure is too high. We reserve the right to decline projects that we consider too risky.

Can we order synthesized aptamers from NeoVentures?

No. There is nothing special about natural oligonucleotides aptamer synthesis. This is the same as primer synthesis for PCR applications. It does not make sense for you to order aptamers from us. We will charge you a higher price than you will pay purchasing the same sequence from an oligonucleotide supplier. We have a relationship with Eurofins in Europe which provides our clients with a discount on synthesis.  Clients are not committed to us for their supply of aptamers that we develop for them.

Are my aptamers ready for commercialization following a project with NeoVentures?

Yes. We perform binding assays under the conditions that you indicate the aptamer will be used commercially. You may wish to contract us to trim the aptamer to a shorter length to reduce production costs. Trimming aptamers improves their performance.

We are prepared to provide you with technical support to your commercial development process. We also offer a full service package where we will do all aspects of aptamer based diagnostic kit development including regulatory approvals.

Why does it take NeoVentures four months to develop an aptamer?

We wish we could do it faster, but our commitment to the development of the best possible aptamer for clients prevents us from doing so. We do not use automated selection processes. All selections are done by hand, results from each selection round are discussed, and dynamic adjustments are made to ensure that we are maintaining appropriate stringency on affinity and often more importantly on specificity.

For next generation sequencing we are currently relying on the use of an Illumina HiSeq 2500 at the Hospital for Sick Children in Toronto. Errors and bias in the next generation sequencing process are poorly understood, especially for aptamer discovery. We do not have a genomic template to compare sequences against. We have developed a database of sequences across a broad array of projects. This enables us to perform meta-analysis to remove NGS bias and to identify what sequences are special to your project. At the end of a project with us you have an aptamer that you can synthesize on a commercial scale in a week, locally. There is no need to clone genes, and develop recombinant expression systems. Aptamer development from project initiation to commercialization is faster than antibody development.

Do aptamers work in the same applications as antibodies?

Anything that an antibody can do an aptamer can do. You will note, I did not say necessarily better. Aptamers can be applied to a broader range of targets than antibodies. We develop aptamers for targets that are too large for antibodies, such as membrane proteins in situ within cells, and targets that are too small for antibodies. It is also possible to develop aptamers for targets that are too toxic for antibody production, where the injection of the target molecule would kill the animal it is being injected into.

Does NeoVentures provide sequence information from the apatmers they develop for clients?

We consider all of the information that we develop within a project as the property of the client. After we receive final payment for a project we release as many sequences to you as you want. With the next generation sequencing that we provide there can be as many as thirty million sequences.

Do your aptamers require denaturing prior to use?

We perform binding assays without denaturing the aptamers first. This ensures that we provide you with aptamers that do not need denaturing first.

What does an integrated Next Generation Sequencing (NGS) strategy mean and why is it important?

At NeoVentures we have pioneered the use of multichannel comparisons for NGS analysis.  Routinely we establish separate selection channels with positive and negative targets. We NGS both channels and compare the rates of enrichment across the channels. This approach identifies aptamers at the sequencing stage that are truly specific to their targets. We also include a negative, no target selection channel. We have found that certain sequences in every selection are enriched as a result of the process in a manner that is not dependent on the target.

What binding assays do NeoVentures use to determine binding affinities?

If the targets are large enough we use our surface plasmon resonance imaging (SPRi) instrument. We have a Horiba OpenPlex instrument that allows us to simultaneously observed binding on multiple aptamers immobilized on a gold chip.

Targets with low mass to be detected by SPRi, are analyzed with isothermal titration calorimetry (ITC). We have a TA Instruments NanoITC with a receiving chamber of 170 uL.

For analysis of aptamer binding in matrix we immobilize the aptamer on resin, flow the target through, and determine the amount bound through HPLC analysis. With protein targets we OPA derivatize in order to enhance sensitivity.

We have also worked extensively with fluorescence polarization and have capacity to analyze binding with this approach with our Tecan Sapphire II instrument.

What makes a target a good target for an aptamer?

The first constraint is aqueous solubility. Many small molecules and peptides are simply not very soluble in aqueous solutions. This means we need to find a balance between the minimum amount of organic solvent that we can use while still maintaining aptamer functionality.

Another constraint is the potential for the target to present itself in multiple forms to the aptamer, or in forms in vitro that are different from forms in vivo.

A third constraint is target stability. Targets that self metabolize or are not stable for more than 1 hour at RT are difficult to work with.

A fourth constraint is the cost of the target. This is often a constraint with proteins where the native form of the protein is necessary in order to obtain epitopes that are present in vivo.

A fifth constraint is target purity. We have failed with projects based on post-translational modifications because the target material was a mixture of PTMs and non-PTM targets. We need purity of at least one side of a contrast, it does not matter if it is the positive or negative side.

Does NeoVentures work with whole cells?

Yes, we have performed several successful projects with whole cells. We prefer targeting the extra-cellular domain of transmembrane proteins directly but this is not always possible. This is the only domain on such proteins that is generally soluble.

Can NeoVentures target epitopes on proteins?

Yes, we prefer to use peptides for selection rather than whole proteins.

How do you know that the epitope from the peptide will be conserved at the whole protein level?

We only use peptides as a guide for selection, we always introduce double positives with peptides and proteins. Also, we only use peptides for which an antibody has previously been shown to bind to as well as the whole protein.