Frequently Asked Questions
Aptamers are RNA or DNA oligonucleotides that bind to specific target molecules with high affinity and specificity. The binding of aptamers to such targets is based on the same mode of action as antibodies, primarily Van der Waals forces and hydrogen bonds. The only difference is that nucleotides are involved in these bonds instead of amino acids. They have similar affinities as antibodies for their targets and provide several advantages, including greater stability, easier large-scale production, low immunogenicity, and the ability to target molecules with low antigenicity. Like antibodies, aptamers have a broad range of applications, serving as drugs, diagnostic and therapeutic tools, analytic reagents, and bio-imaging molecules.
The classic design of an aptamer library is provided in the following figure.
There is a random sequence of usually 40 nucleotides (nt) that is flanked by two PCR primer recognition sequences. There are two factors that enable aptamers to work, one is that this is a single stranded oligonucleotide. This means that each sequence wants to be as double stranded as possible. Each sequence will fold back on itself in regions of homology (A pairing with T, and G pairing with C) as much as possible. The second factor that enables aptamers to work is the presence of a random region. When an aptamer library is synthesized there is an equal probability of every nucleotide at every position in the random region. Every different sequence will fold back in a different way, thus forming a different shape and thus exhibiting potential for binding to different molecules. The challenge with aptamer selection is to find the sequences that bind to your target molecule.
SELEX is the process by which aptamers that bind to a target are selected from a library of random sequences. The process involves exposing a random library to the immobilized target, partitioning bound aptamers from unbound ones, PCR amplifying the bound aptamers, and repeating the process. This process is repeated until the desired aptamers dominate the selection library.
The key constraint with SELEX is the need to immobilize the target in order to partition bound aptamers from unbound ones. This is a problem with small molecules because the immobilization both changes the structure of the molecule and removes a charge group from selection for binding. FRELEX overcomes this constraint by enabling partitioning of bound aptamers from unbound ones in a free state. Learn more about the advantages of FRELEX here
From the data, DNA aptamer works 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. However, we are just beginning to scratch the surface regarding what tertiary structures single stranded DNA molecules can form. G-quartet structures make excellent aptamers with binding affinities like 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.
For diagnostic applications there is currently no 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.
Anything that an antibody can do an aptamer can do. 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. Learn more here.
The dissociation constant (kD) is a measure of the binding strength between the aptamer and target. A low value for the dissociation constant means a low concentration of aptamer and target are required for binding to occur and indicates high affinity or binding strength. kD = kd/ka, or koff/kon. In diagnostic applications the lower the koff the better because this means that your complex will stay together in wash steps.
- Soluble in aqueous solutions: 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.
- A stable form of the target: If a target presents itself in multiple forms to the aptamer, or in forms in vitro that are different from forms in vivo, this creates an issue for selection as the epitopes constantly change
- Target is stabile in relevant matrix: Targets that self metabolizes or are not stable for more than 1 hour at RT are difficult to work with.
- Low 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.
- High 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 a pure sample of at least one side of a contrast, it does not matter if it is the positive or negative side
There are several simple reasons:
- Aptamer based selection does not have an implicit means of removing sequences that bind to self targets (the auto-immune system of antibody production). Counter selection is not sufficiently effective to overcome this constraint. NeoVentures has developed the Neomer approach to overcome this problem and develop aptamers that are more suitable for commercial diagnostic applications than ever before.
- 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 aptamer 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.
Aptamers can be used in the same diagnostic platforms that antibodies can be used in, for example, Lateral flow, flow-through, fluorescent /colorimetric plate-based tests, electrochemistry, and qPCR tests. In certain circumstances, aptamers will need different modifications compared to what is used for antibodies to be functional. For example, aptamers can be used in lateral flow-based tests, but they need to be functionalized prior to being put onto the nitrocellulose pad or else they will not be functional to bind to their target. At NeoVentures, we know what chemistry works the best for this diagnostic test, as well as all of the other platforms.
A novel method of aptamer selection, Neomers, eliminates the key problems facing aptamer development observed while using SELEX. It uses a combination of known and random nucleotides that allows for the use of a library of the same 4.29 billion sequences for every selection. We can characterize the level of binding of a target molecule on all 4.29 billion sequences in a single selection round, followed by a clever trick we employ for NGS analysis. Read more about Neomers here.
Neomers overcomes all the problems SELEX has. Due to the novel way that the library is designed, we can start with the same library for every selection. Using this method, we can account for those sequences that bind to the off targets, even those that bind weakly, and remove them from the list of potential sequences his allows us to create an ‘immune tolerance’ system in-silico and remove any sequences that bind to a ‘self’ antigen.
There are several reasons that the Neomer approach represents a
generational shift in aptamer technology. All of these reasons arise from
the capacity to evaluate the response of the same 4.29 billion sequences
against different targets.
Increased speed of development (one round of selection)
Reproducible selection results (start with the same library on the
same target in different replications)Building knowledge base (every new target adds to the known response
fingerprint of the 4.29 billion sequences).Increased specificity (ability to screen all sequences that bind to
desired target, for their response to abundant proteins)
It should be recalled that the SELEX platform was proprietary and no
commercial products were developed without a need for a license with the
patent holders. NeoVentures is not taking this approach. We are licensing
the platform to others to use, and we do not require a royalty for Neomer
based aptamers.
The platform requires intensive computing capacity. We have built
proprietary code to perform analyses on in-house Linux servers and as part
of the licensed use of the platforms it is necessary that we perform the
data analysis ourselves. The selection process and the NGS processing of
libraries can be done by others.
Since our invention of the Neomer platform we have been have experienced a
steady increase in demand for aptamers from new clients and from our large
base of existing clients. Our clients come first, and our results with the
clients are maintained as confidential. We have continued to increase our
staff steadily over the last two years and we are now processing results
from in-house work for publication.
If you have the capacity for selection in-house, then this route offers substantial savings over our traditional custom aptamer design. NeoVentures would provide the Neomer library for a selection that you would complete. There is the option of either you complete the NGS preparation and NGS submission, or NeoVentures can for an additional cost. After NGS, NeoVentures would analyze the data the same way that we do for any of our in-house projects to get you the highest-quality aptamers and structure analysis. Learn more about our licensing options here.
We have selected and tested aptamers for a variety of targets. These are available to purchase for feasibility testing in various assays. Additionally, there is the option for exclusive commercial rights for an additional fee.
No, we think our competition is with antibody providers. Antibody providers do not take an IP position on antibodies that are custom developed for you. 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.
These answers tend to be biased to favor the aptamer company. To maintain transparency, NeoVentures structures 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.
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.
Yes, we prefer to use peptides for selection rather than whole proteins. 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.
- – Large targets (i.e. proteins): Surface plasmon resonance imaging (SPRi) instrument. We have a Horiba OpenPlex instrument that allows us to simultaneously observe binding on multiple aptamers immobilized on a gold chip.
- – Small targets: Isothermal titration calorimetry (ITC). We have a TA Instruments NanoITC.
- – Analysis of aptamer binding in matrix: we immobilize the aptamer on resin, flow the target through, and determine the amount bound through HPLC analysis.
Additionally, we have worked extensively with fluorescence polarization and fluorescence switch assays and have capacity to analyze binding with these approaches with our Tecan Sapphire II instrument.
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.
Yes, for most targets NeoVentures can test in our binding assays to advise on a pair of aptamers that have the best chance of being a successful sandwich pair. These are typically only feasible for protein or large peptide targets, as there will be multiple epitopes available for a pair of aptamers binding.
We perform binding assays without denaturing the aptamers first. This ensures that we provide you with aptamers that do not need denaturing first. Learn why in our blog.
Aptamers are RNA or DNA oligonucleotides that bind to specific target molecules with high affinity and specificity. The binding of aptamers to such targets is based on the same mode of action as antibodies, primarily Van der Waals forces and hydrogen bonds. The only difference is that nucleotides are involved in these bonds instead of amino acids. They have similar affinities as antibodies for their targets and provide several advantages, including greater stability, easier large-scale production, low immunogenicity, and the ability to target molecules with low antigenicity. Like antibodies, aptamers have a broad range of applications, serving as drugs, diagnostic and therapeutic tools, analytic reagents, and bio-imaging molecules.
The classic design of an aptamer library is provided in the following figure.
There is a random sequence of usually 40 nucleotides (nt) that is flanked by two PCR primer recognition sequences. There are two factors that enable aptamers to work, one is that this is a single stranded oligonucleotide. This means that each sequence wants to be as double stranded as possible. Each sequence will fold back on itself in regions of homology (A pairing with T, and G pairing with C) as much as possible. The second factor that enables aptamers to work is the presence of a random region. When an aptamer library is synthesized there is an equal probability of every nucleotide at every position in the random region. Every different sequence will fold back in a different way, thus forming a different shape and thus exhibiting potential for binding to different molecules. The challenge with aptamer selection is to find the sequences that bind to your target molecule.
SELEX is the process by which aptamers that bind to a target are selected from a library of random sequences. The process involves exposing a random library to the immobilized target, partitioning bound aptamers from unbound ones, PCR amplifying the bound aptamers, and repeating the process. This process is repeated until the desired aptamers dominate the selection library.
The key constraint with SELEX is the need to immobilize the target in order to partition bound aptamers from unbound ones. This is a problem with small molecules because the immobilization both changes the structure of the molecule and removes a charge group from selection for binding. FRELEX overcomes this constraint by enabling partitioning of bound aptamers from unbound ones in a free state. Learn more about the advantages of FRELEX here
From the data, DNA aptamer works 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. However, we are just beginning to scratch the surface regarding what tertiary structures single stranded DNA molecules can form. G-quartet structures make excellent aptamers with binding affinities like 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.
For diagnostic applications there is currently no 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.
Anything that an antibody can do an aptamer can do. 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. Learn more here.
The dissociation constant (kD) is a measure of the binding strength between the aptamer and target. A low value for the dissociation constant means a low concentration of aptamer and target are required for binding to occur and indicates high affinity or binding strength. kD = kd/ka, or koff/kon. In diagnostic applications the lower the koff the better because this means that your complex will stay together in wash steps.
1. Soluble in aqueous solutions: 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.
2. A stable form of the target: If a target presents itself in multiple forms to the aptamer, or in forms in vitro that are different from forms in vivo, this creates an issue for selection as the epitopes constantly change
3. Target is stabile in relevant matrix: Targets that self metabolizes or are not stable for more than 1 hour at RT are difficult to work with.
4. Low 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.
5. High 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 a pure sample of at least one side of a contrast, it does not matter if it is the positive or negative side
There are several simple reasons:
- Aptamer based selection does not have an implicit means of removing sequences that bind to self targets (the auto-immune system of antibody production). Counter selection is not sufficiently effective to overcome this constraint. NeoVentures has developed the Neomer approach to overcome this problem and develop aptamers that are more suitable for commercial diagnostic applications than ever before.
- 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 aptamer 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.
Aptamers can be used in the same diagnostic platforms that antibodies can be used in, for example, Lateral flow, flow-through, fluorescent /colorimetric plate-based tests, electrochemistry, and qPCR tests. In certain circumstances, aptamers will need different modifications compared to what is used for antibodies to be functional. For example, aptamers can be used in lateral flow-based tests, but they need to be functionalized prior to being put onto the nitrocellulose pad or else they will not be functional to bind to their target. At NeoVentures, we know what chemistry works the best for this diagnostic test, as well as all of the other platforms.
A novel method of aptamer selection, Neomers, eliminates the key problems facing aptamer development observed while using SELEX. It uses a combination of known and random nucleotides that allows for the use of a library of the same 4.29 billion sequences for every selection. We can characterize the level of binding of a target molecule on all 4.29 billion sequences in a single selection round, followed by a clever trick we employ for NGS analysis. Read more about Neomers here.
Neomers overcomes all the problems SELEX has. Due to the novel way that the library is designed, we can start with the same library for every selection. Using this method, we can account for those sequences that bind to the off targets, even those that bind weakly, and remove them from the list of potential sequences his allows us to create an ‘immune tolerance’ system in-silico and remove any sequences that bind to a ‘self’ antigen.
If you have the capacity for selection in-house, then this route offers substantial savings over our traditional custom aptamer design. NeoVentures would provide the Neomer library for a selection that you would complete. There is the option of either you complete the NGS preparation and NGS submission, or NeoVentures can for an additional cost. After NGS, NeoVentures would analyze the data the same way that we do for any of our in-house projects to get you the highest-quality aptamers and structure analysis. Learn more about our licensing options here.
We have selected and tested aptamers for a variety of targets. These are available to purchase for feasibility testing in various assays. Additionally, there is the option for exclusive commercial rights for an additional fee.
No, we think our competition is with antibody providers. Antibody providers do not take an IP position on antibodies that are custom developed for you. 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.
These answers tend to be biased to favor the aptamer company. To maintain transparency, NeoVentures structures 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.
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.
Yes, we prefer to use peptides for selection rather than whole proteins. 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.
- – Large targets (i.e. proteins): Surface plasmon resonance imaging (SPRi) instrument. We have a Horiba OpenPlex instrument that allows us to simultaneously observe binding on multiple aptamers immobilized on a gold chip.
- – Small targets: Isothermal titration calorimetry (ITC). We have a TA Instruments NanoITC.
- – Analysis of aptamer binding in matrix: we immobilize the aptamer on resin, flow the target through, and determine the amount bound through HPLC analysis.
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.
Yes, for most targets NeoVentures can test in our binding assays to advise on a pair of aptamers that have the best chance of being a successful sandwich pair. These are typically only feasible for protein or large peptide targets, as there will be multiple epitopes available for a pair of aptamers binding.
We perform binding assays without denaturing the aptamers first. This ensures that we provide you with aptamers that do not need denaturing first. Learn why in our blog.
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