{"id":4806,"date":"2023-08-07T10:59:33","date_gmt":"2023-08-07T14:59:33","guid":{"rendered":"https:\/\/neoaptamers.com\/?page_id=4806"},"modified":"2024-12-04T11:04:52","modified_gmt":"2024-12-04T16:04:52","slug":"neomers-approach","status":"publish","type":"page","link":"https:\/\/neoaptamers.com\/fr\/neomers-approach\/","title":{"rendered":"Neomers – AUG UPDATES"},"content":{"rendered":"\t\t
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Neomers<\/h1>\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t
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An innovative new approach to aptamer selection that overcomes the problems associated with SELEX.\u00a0 <\/strong>Explore what makes Neomers unique now, or get in touch with our team directly to find out how it can benefit your unique application.<\/b><\/h4>\t\t\t\t\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t
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\n\t\t\t\t\t\n\t\t\t\t\t\t\n\t\t\t\t\t\t\t\t\tCONTACT US<\/span>\n\t\t\t\t\t<\/span>\n\t\t\t\t\t<\/a>\n\t\t\t\t<\/div>\n\t\t\t\t\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t\t<\/div>\n\t\t<\/div>\n\t\t\t\t\t<\/div>\n\t\t<\/section>\n\t\t\t\t
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What is SELEX?<\/h2>\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t
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SELEX<\/b> stands for S<\/b>ystematic E<\/b>volution of L<\/b>igands by EX<\/b>ponential enrichment. It is a technique used to identify and isolate specific nucleic acid molecules, such as DNA or RNA aptamers, that can bind to a target molecule with high affinity and specificity.<\/p>

The SELEX process involves several iterative rounds of selection and amplification, where a diverse pool of nucleic acid molecules is exposed to the target molecule. After each round, the bound nucleic acid molecules are separated from the unbound ones, and the bound molecules are then amplified through polymerase chain reaction (PCR) or reverse transcription PCR (RT-PCR) for RNA-based SELEX.<\/p>

During subsequent rounds, the selection conditions are made more stringent to enrich for the nucleic
acid molecules that have the highest affinity for the target. As the process continues, the pool of
aptamers becomes increasingly enriched for those that can effectively bind to the target, leading to the isolation of specific aptamers with strong binding affinities.<\/p>\t\t\t\t\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t

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What is SELEX?<\/h3>\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t
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SELEX stands for Systematic Evolution of Ligands by EXponential enrichment. It is a technique used to identify and isolate specific nucleic acid molecules, such as DNA or RNA aptamers, that can bind to a target molecule with high affinity and specificity.<\/p>

The SELEX process involves several iterative rounds of selection and amplification, where a diverse pool of nucleic acid molecules is exposed to the target molecule. After each round, the bound nucleic acid molecules are separated from the unbound ones, and the bound molecules are then amplified through polymerase chain reaction (PCR) or reverse transcription PCR (RT-PCR) for RNA-based SELEX.<\/p>

During subsequent rounds, the selection conditions are made more stringent to enrich for the nucleic
acid molecules that have the highest affinity for the target. As the process continues, the pool of
aptamers becomes increasingly enriched for those that can effectively bind to the target, leading to the isolation of specific aptamers with strong binding affinities.<\/p>\t\t\t\t\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t

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The Problem with SELEX is the Random Region.<\/h2>\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t
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\u00a0The following figure shows a classic SELEX aptamer selection library.\u00a0<\/p>\t\t\t\t\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t

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The random sequence in the initial library is composed of 40 nucleotides, meaning that there are 1.24 E24 possible sequences. The contiguous nature of the random region imposes a constraint on structural diversity. There is a need for at least three nucleotides to form a hairpin. As such the random sequence has to be 40 nt in order to deliver an adequate coverage of structural diversity. SELEX starts with an aliquot of the synthesized random library representing 1E15 sequences. This is the maximum number of sequences that can be applied experimentally. This represents a very small subset of the possible 1.2E24 sequences in the synthesized library resulting in two constraints.<\/p>

1. Every selection is started with completely different sequences.<\/p>

2. Each sequence in the starting library is present on average as a single copy.\u00a0<\/p>

Starting selection with a single copy of each sequence means that several rounds of reiterative selection must be performed in order to enrich the copy number of those sequences that bind to the target. This sounds fine, but starting with a single copy of each sequence also means that the majority of the sequences are lost arbitrarily (whether they bind or not) in the first selection round.\u00a0<\/p>

1E15 sequences is such a small subsample of the possible sequences that it is a given that there will be virtually no overlap of sequences from one aliquot of library to another. As such, it is not possible to replicate selection of the same sequences on different targets. We and others have attempted to overcome this problem by using negative selection against counter targets where we discard sequences that bind to other targets. This works well for sequences that cross-react strongly, but this does not work well for weakly cross-reacting sequences.\u00a0\u00a0\u00a0<\/p>\t\t\t\t\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t

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The Problem with SELEX is the Random Region. <\/h3>\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t
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\u00a0The following figure shows a classic SELEX aptamer selection library.\u00a0<\/p>\t\t\t\t\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t

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The random sequence in the initial library is composed of 40 nucleotides, meaning that there are 1.24 E24 possible sequences. The contiguous nature of the random region imposes a constraint on structural diversity. There is a need for at least three nucleotides to form a hairpin. As such the random sequence has to be 40 nt in order to deliver an adequate coverage of structural diversity. SELEX starts with an aliquot of the synthesized random library representing 1E15 sequences. This is the maximum number of sequences that can be applied experimentally. This represents a very small subset of the possible 1.2E24 sequences in the synthesized library resulting in two constraints.<\/p>

1. Every selection is started with completely different sequences.<\/p>

2. Each sequence in the starting library is present on average as a single copy.\u00a0<\/p>

Starting selection with a single copy of each sequence means that several rounds of reiterative selection must be performed in order to enrich the copy number of those sequences that bind to the target. This sounds fine, but starting with a single copy of each sequence also means that the majority of the sequences are lost arbitrarily (whether they bind or not) in the first selection round.\u00a0<\/p>

1E15 sequences is such a small subsample of the possible sequences that it is a given that there will be virtually no overlap of sequences from one aliquot of library to another. As such, it is not possible to replicate selection of the same sequences on different targets. We and others have attempted to overcome this problem by using negative selection against counter targets where we discard sequences that bind to other targets. This works well for sequences that cross-react strongly, but this does not work well for weakly cross-reacting sequences.\u00a0\u00a0\u00a0<\/p>\t\t\t\t\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t

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Why is this Really a Problem?<\/h2>\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t
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Abundant proteins in blood like human serum albumin are present at a concentration of 600 uM. If your target is present in blood at a concentration of 600 pM, this is a million fold difference.\u00a0 Aptamers need to have a million fold greater affinity for your target than for HSA. Antibodies have this, as a result of immune tolerance in their development. It is not possible to obtain this level of specificity with counter selection with aptamers. This is why aptamers have worked well in publications against targets in buffer, but not well against the same targets in complex matrices represented by biological fluids.<\/p>\t\t\t\t\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t\t<\/div>\n\t\t<\/div>\n\t\t\t\t

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Why is this Really a Problem?<\/h3>\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t
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Abundant proteins in blood like human serum albumin are present at a concentration of 600 uM. If your target is present in blood at a concentration of 600 pM, this is a million fold difference.\u00a0 Aptamers need to have a million fold greater affinity for your target than for HSA. Antibodies have this, as a result of immune tolerance in their development. It is not possible to obtain this level of specificity with counter selection with aptamers. This is why aptamers have worked well in publications against targets in buffer, but not well against the same targets in complex matrices represented by biological fluids.<\/p>\t\t\t\t\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t\t<\/div>\n\t\t<\/div>\n\t\t\t\t

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This is why Aptamers Have Failed to Date in Commercial Applications<\/h2>\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t
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The Neomer method overcomes these constraints:<\/b><\/p>

We designed the Neomer selection method to overcome the difficulties implicit with SELEX. One core insight that enables the Neomer method was that it was possible to generate a library of sequences that maintains the same level of structural diversity as a contiguous 40 nt random region in a SELEX library by interspersing sixteen random nucleotides in between fixed sequences. The fixed sequences in our libraries are designed such that they do not hybridize with each other. Secondary structure in the library is driven by the identity of the non-contiguous random nucleotides. A library with 14 random nucleotides consists of 2.68E8 possible sequences. We start selection with these libraries with 2.68E12 sequences, thus obtaining an average of 10,000 copies of each sequence at the start of selection. This eliminates the risk of arbitrarily losing good sequences in the first round of selection. This also enables us to characterize the effect of selection in a single selection round.\u00a0<\/p>\t\t\t\t\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t

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This is why Aptamers Have Failed to Date in Commercial Applications<\/h3>\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t
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The Neomer method overcomes these constraints:<\/b><\/p>

We designed the Neomer selection method to overcome the difficulties implicit with SELEX. One core insight that enables the Neomer method was that it was possible to generate a library of sequences that maintains the same level of structural diversity as a contiguous 40 nt random region in a SELEX library by interspersing sixteen random nucleotides in between fixed sequences. The fixed sequences in our libraries are designed such that they do not hybridize with each other. Secondary structure in the library is driven by the identity of the non-contiguous random nucleotides. A library with 16 random nucleotides consists of 4.29E9 possible sequences. We start selection with these libraries with 4.29E12 sequences, thus obtaining an average of 1,000 copies of each sequence at the start of selection. This eliminates the risk of arbitrarily losing good sequences in the first round of selection. This also enables us to characterize the effect of selection in a single selection round.\u00a0<\/p>\t\t\t\t\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t

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Transforming Aptamer Development from being an Art to\nScience<\/h2>\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t
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We have detailed how a library of 2.68E8 unique sequences can be applied across various targets, enabling us to apply the same starting library to each target with only a single round of selection. In practice, we conduct selection in triplicate for each target as well as for a negative control. By assessing the counts of each unique sequence, we can explore the structure space to identify which structures show enrichment within the positive target library.<\/p>

The Neomer approach leverages advanced statistical methods to improve the efficiency and reliability of the selection process and select the top performing structures from the selected library. Central to this analysis are the enrichment metrics: target and counter-target averages, log-transformed ratios, and other statistical metrics such as p-values. These parameters allow for a nuanced assessment of aptamer specificity and performance across various selection conditions. By comparing these metrics between different selections, we can identify structures that demonstrate consistent enrichment for the target of interest while maintaining low affinity for counter-targets, achieving unprecedented levels of specificity. An example would include screening against common-counter targets such as abundant blood proteins like HSA and IgG. Further steps are then taken to characterize sequences enriched per selected structure.\u00a0This approach creates an in silico immune tolerance system for aptamer development. Such specificity is critical for the success of aptamers in commercial applications.<\/p>

The Neomer approach represents the next generation of aptamer development, elevating it from an empirical process to a scientifically rigorous and highly precise methodology.<\/p>\t\t\t\t\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t\t<\/div>\n\t\t<\/div>\n\t\t\t\t\t<\/div>\n\t\t<\/section>\n\t\t\t\t

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A second key insight was the development of a process that would enable us to characterize all of the 4.29E9 sequences in a single next generation sequencing analysis. We obtain an average of 10 to 20 million reads per library with Illumina NovaSeq sequencing. This is not sufficient to allow coverage of all library sequences. To overcome this constraint, we have built a restriction site into the middle of the library. After selection, and amplification, we use this site to cut the library into two parts (modules) each containing eight random nucleotides. Each module consists of 65,536 possible sequences. We obtain coverage of all possible sequences in each module with 10 million reads in NGS. Then, we multiply the frequencies observed in module A with those observed in module B for each selected library. This out product matrix or Punnett square contains the frequencies of all 4.29E9 sequences. <\/p>

\u00a0<\/p>\t\t\t\t\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t\t<\/div>\n\t\t<\/div>\n\t\t\t\t

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Moving Aptamer Development to Being a Science<\/h3>\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t
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We have explained how we can apply the same set of 4.29E9 sequences to different targets and
characterize the effect of a single round of selection on each sequence. In practice, we use this capacity to perform a selection on each target in triplicate and on a negative control in triplicate. We have developed software on our own Linux servers that enable us to determine the averages and the standard deviations of these averages for each of the 4.29E9 sequences for each treatment. We then compare the positive averages to the negative averages and develop a 4.29E9 matrix of Z values. Our software outputs the top 10,000 sequences based on Z score.<\/p>\t\t\t\t\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t

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We have also developed software that enables us to screen for the performance of the top 10,000
sequences selected for your target in selection against other targets such as abundant proteins in blood like HSA and IgG. We filter the sequences that bind well to your target and select as candidates only those that exhibit no response to these counter targets. In this way we are able to obtain unprecedented levels of specificity. In effect we have created an in silico immune tolerance system for aptamer development. This level of specificity is necessary for aptamers to be successful in commercial applications. That is why we consider the Neomer approach as the next level and the future generation of all aptamer development.<\/p>\t\t\t\t\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t\t<\/div>\n\t\t<\/div>\n\t\t\t\t\t<\/div>\n\t\t<\/section>\n\t\t\t\t

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In Silico Aptamer Selection Options\n\n<\/h2>\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t
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NeoVentures now provides additional project options with\nIn Silico aptamer selection, offering significant cost savings compared to\nour traditional custom aptamer design process. Learn more about our In\nSilico solutions today!<\/o:p><\/p>\t\t\t\t\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t

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Licensing Opportunities<\/h2>\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t
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We have decided to make the Neomer library available for licensing to labs that have the capacity for selection in-house. This route offers substantial savings over our traditional custom aptamer design. Find out more about licensing.<\/p>\t\t\t\t\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t

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Revolutionize your Business with Neomers<\/h2>\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t
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Our Process<\/h3>\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t
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\n\t\t\t\t\t\t\n\t\t\t\t\t\t\tFree Consultation\t\t\t\t\t\t<\/a>\n\t\t\t\t\t<\/h3>\n\t\t\t\t\n\t\t\t\t\n\t\t\t<\/div>\n\t\t\t\n\t\t<\/div>\n\t\t\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t\t<\/div>\n\t\t<\/div>\n\t\t\t\t
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\n\t\t\t\t\t\t\n\t\t\t\t\t\t\tCustomize a Strategy\t\t\t\t\t\t<\/a>\n\t\t\t\t\t<\/h3>\n\t\t\t\t\n\t\t\t\t\n\t\t\t<\/div>\n\t\t\t\n\t\t<\/div>\n\t\t\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t\t<\/div>\n\t\t<\/div>\n\t\t\t\t\t<\/div>\n\t\t<\/section>\n\t\t\t\t
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\n\t\t\t\t\t\t\n\t\t\t\t\t\t\tWe Develop It\t\t\t\t\t\t<\/a>\n\t\t\t\t\t<\/h3>\n\t\t\t\t\n\t\t\t\t\n\t\t\t<\/div>\n\t\t\t\n\t\t<\/div>\n\t\t\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t\t<\/div>\n\t\t<\/div>\n\t\t\t\t
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\n\t\t\t\t\t\t\n\t\t\t\t\t\t\tOperational Aptamers\t\t\t\t\t\t<\/a>\n\t\t\t\t\t<\/h3>\n\t\t\t\t\n\t\t\t\t\n\t\t\t<\/div>\n\t\t\t\n\t\t<\/div>\n\t\t\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t\t<\/div>\n\t\t<\/div>\n\t\t\t\t\t<\/div>\n\t\t<\/section>\n\t\t\t\t\t<\/div>\n\t\t<\/div>\n\t\t\t\t
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Start Now with a Free Consultation<\/h2>\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t
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