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Planning to work with aptamers?

IDT synthesizes aptamer libraries

We are often asked whether IDT manufactures aptamers. The answer is, yes! IDT does synthesize nucleic acid aptamers and aptamer libraries, and there are already 100s of published research papers describing the successful use of such sequences manufactured by IDT (see the sidebar, Selected aptamer citations, below). IDT scientists also collaborate with outside research groups on aptamer design and aptamer applications (for a list of these publications, visit IDT publications—Aptamers).

You must provide the aptamer designs

However, IDT cannot design an aptamer library for you. You will need to provide the sequences for synthesis. Aptamer sequences should be based on the results of aptamer selection technology, such as SELEX (Systematic Evolution of Ligands by Exponential enrichment; see the sidebar, What is SELEX?, below).

Length limits for aptamers

Most aptamers are 20–80 nt, single-stranded DNA or RNA sequences. However, IDT can synthesize longer aptamers, if needed.

Definition: Aptamers

Aptamers are short (20–80mer), single-stranded DNA or RNA sequences or proteins that bind to target molecules with high affinity and specificity through their 3-dimensional structures. RNA sequences make up the majority of nucleic acid aptamers, perhaps because they can be synthesized by in vitro transcription in the laboratory and, with a 2′-OH, would potentially provide more diverse secondary structure than single-stranded DNA molecules. Nucleic acid aptamers are often identified using an iterative enrichment technique, where oligos or proteins with increased affinity and specificity to a target molecule are isolated from a sequence pool after several rounds of selection. Nucleic acid aptamers are selected in vitro based on affinity for the target molecule, which might be a protein, virus, or cell. SELEX (Systematic Evolution of Ligands by EXponential enrichment; see the sidebar, What is SELEX?) is one of the most common iterative enrichment methods used to Identify nucleic acid aptamers.


Figure 1. Engineering aptamers to bind specific targets.

Because they have similar target binding affinities for their targets as antibodies, yet offer several advantages over antibody-based affinity molecules, aptamers are often used as substitutes for antibodies. Aptamers are typically easier to produce, especially on a large scale. They are physically more stable, and modifications that increase intracellular stability are easily incorporated, all at a lower cost. Aptamers are also easily purified and typically have low immunogenicity. They penetrate tissues to reach their target sites faster and more effectively than antibodies, due to their smaller size (8–25 kDa nucleic acids vs. ~150 kDa antibodies), and are also able to target molecules with low antigenicity; for example, when protein targets might otherwise not provide enough epitopes for antibody binding because they are fragmented or denatured [1,2].

Like antibodies, aptamers have a broad range of applications, serving as drugs, diagnostic tools, analytic reagents, bio-imaging molecules, and biosensors (aptasensors) [1–5]. Aptamers can also be used in a targeted therapeutic role by delivering nanoparticles, antibodies, and other drugs to cancer cells through conjugation to these molecules [2].

What is SELEX?

SELEX (Systematic Evolution of Ligands by EXponential enrichment) is a recursive nucleic acid aptamer selection technology. It works by isolating sequences with increased affinity and specificity to a target molecule from an oligo sequence pool through several rounds of selection. The process proceeds as follows (also, see Figure 2):

  • A library of single-stranded DNA or RNA is generated. These sequences consist of a variable sequence region (usually 30–40 nt) flanked by static binding sites on either side.
  • The library is incubated with the selected target molecule. DNA or RNA molecules that bind to the target are prospective aptamers for that target. After unbound sequences are filtered out, the bound sequences are separated from the target and purified. Nitrocellulose membrane filtration, affinity chromatography/magnetic bead, capillary electrophoresis, microfluidic chips, atomic force microscopy (AFM), electrophoretic mobility shift assays (EMSA), and surface plasmon resonance (SPR) are some of the technologies used to separate bound from unbound sequences.
  • The bound sequences are then PCR amplified, creating a more specific sequence library. This library is used in a new round of SELEX to further optimize the quality of the aptamers.

Figure 2. Iterative selection of target specific nucleic acids using SELEX.

The SELEX procedure can fail to produce promising aptamers due to: inadequate library design, non-specific sequence retention, accumulation of amplification artifacts, and use of screening criteria that are not associated with actual application readouts [6]. With the wide range of applications for aptamers, and the limitations of SELEX, improvements and alternatives to SELEX continue to arise (e.g., High-Fidelity SELEX [6], and MAWS—Making Aptamers Without SELEX [7]).

Base modifications increase aptamer options

Base modifications can be added to aptamers for purification (e.g., 5′-biotin), for detection (e.g., 6-FAM), and to enhance stability during in vitro and in vivo use (e.g., 2′-O-Methyl RNA bases or 2′-Fluoro bases) [8,9]. You must provide IDT with your sequence designs, including specifying modifications and positions, for synthesis.

Appropriate modifications and their positioning should be determined experimentally for optimal aptamer function. As an easy way to generate aptamer designs in the laboratory for initial testing, scientists will sometimes in vitro transcribe RNA aptamers. When doing this, they often include 2′-Fluoro pyrimidine base modifications to improve aptamer stability. Note, however, that these in vitro transcribed molecules will have 2′-Fluoro modifications on every pyrimidine in the sequence. RNA oligos with so many 2′‑Fluoro base insertions may not lead to the most effective aptamers. Furthermore, when the researcher turns to an oligo manufacturer to provide scaled up aptamer yields, such heavily modified aptamers can prove challenging to chemically synthesize (not to mention expensive). In fact, IDT limits standard RNA oligo synthesis to 20 or fewer 2′-Fluoro base modifications (sequence designs with >20, 2′-Fluoro bases can be submitted for special consideration by contacting noncat@idtdna.com, and we frequently receive such requests). Thus, in vitro transcription may not always prove to be the best way to generate modified oligos for testing aptamer function.

Unlike 2′-Fluoro pyrimidine, other common aptamer modifications are more difficult to produce in the lab. For example, IDT can incorporate 2′-Fluoro purines, 2′-O-Methyl, FAM, and biotin modifications into your sequence. These modifications and their positioning would also need to be tested to determine their effect on aptamer structure and function. However, there is currently no easy way to produce these molecules within most labs, and it can get expensive to have multiple variants synthesized by IDT.

Purification recommended for fixed aptamer sequences

If you are ordering aptamers with fixed sequences, IDT recommends HPLC or PAGE purification. For aptamer libraries containing variable bases, IDT recommends standard desalt.

How to order aptamers

To order aptamers online, go to Custom DNA Oligos or Custom RNA Oligos, found on the Order Menu of our website (www.idtdna.com). Enter your desired scale, purification, and the sequence(s) with any random bases or modifications.

For aptamer sequence designs with greater than 20 2′-Fluoro bases inserted, or with modifications not listed on our website, please submit a request for review of your design by emailing noncat@idtdna.com with your name, organization, and sequences. If you would like help with your order, contact our customer service colleagues at custcare@idtdna.com.

Selected aptamer citations

  • Aptekar S, Arora M, et al. (2015) Selective targeting to glioma with nucleic acid aptamers. PloS One, 10(8):e0134957; doi: 10.1371/journal.pone.0134957. The specificity, uptake, and target binding strength of 2 DNA aptamers were investigated in glioma cells and patient tissue. Aptamers were synthesized by IDT and conjugated at the 5' end with either Cy® 3 or with biotin for purification.
  • Bruno JG, Richarte AM. (2015) Development and characterization of an enzyme-linked DNA aptamer-magnetic bead-based assay for human IGF-I in serum. Microchem J, 124:90–95; doi:10.1016/j.microc.2015.08.002. 36 unique DNA aptamers were developed against human IGF-I and evaluated using a unique ELISA-like aptamer-based assay (ELASA). All DNA templates, primers, and biotinylated candidate aptamers were synthesized by IDT.
  • Jacobson O, Yan X, et al. (2015) PET imaging of tenascin-C with a radiolabeled single-strand DNA aptamer. J Nuclear Med 56(4):616-621; doi 10.2967/jnumed.114.149484. Development of the first agent for imaging and quantifying the cancer associated protein, tenascin-C—a tenascin-C-specific single-stranded DNA aptamer. The aptamer was radiolabeled with 18F and 64Cu and used in PET-imaging studies to measure tumor uptake and metabolism. All aptamers, including FITC-aptamers, were synthesized by IDT.
  • Lum J, Wang R, et al. (2015) An impedance aptasensor with microfluidic chips for specific detection of H5N1 avian influenza virus. Sensors, 15: 18565–18578; doi:10.3390/s150818565. Use of a DNA aptamer to replace monoclonal antibodies in an impedance biosensor that uses microfluidics and a microelectrode for detection of the target molecule, in this case, the H5N1 subtype of avian influenza virus. Aptamer secondary structures and delta G were calculated using the free IDT online UNAFold and OligoAnalyzer® programs. Biotinylated aptamers were synthesized by IDT with biotin conjugated at the 5′ end.
  • Moore MD, Escudero-Abarca BI, et al. (2015) Generation and characterization of nucleic acid aptamers targeting the capsid P domain of a human norovirus GII. 4 strain. J Biotechnol, 209:41–49. doi:10.1016/j.jbiotec.2015.06.389. This is the first published account of researchers using a human norovirus protein domain to generate aptamers that recognize and bind diverse norovirus strains. The aptamer library had a 40-nt variable region and was synthesized by IDT.
  • Ouellet E, Foley JH, et al. (2015) Hi‐Fi SELEX: A high‐fidelity digital‐PCR based therapeutic aptamer discovery platform. Biotechnol Bioeng, 112(8):1506–1522; doi: 10.1002/bit.25581. Description of a new aptamer selection platform that uses fixed-region blocking elements to ensure library diversity and guard against amplification artifacts. Truncated (random-region sequence only) and full-length library versions of aptamers were synthesized and HPLC purified by IDT.
  • Takahashi M, Burnett JC, Rossi JJ. (2015) Aptamer-siRNA chimeras for HIV. Adv Exp Med Biol, 848:211–234; doi: 10.1007/978-1-4939-2432-5_11. A review of the use of aptamers to target siRNAs to HIV-1 proteins.
  • Lou X, Qian J, et al. (2009) Micromagnetic selection of aptamers in microfluidic channels.. Proc Natl Acad Sci USA, 106(9):2989–2994; doi: 10.1073/pnas.0813135106. Microfluidic screening of aptamers to the light chain of recombinant Botulinum neurotoxin type A. The DNA library was comprised of sequences containing a central random region of 60 bases flanked by 2 specific 20-base sequences, and was synthesized and purified by IDT.

References

  1. Song K-M, Lee S, Ban C. (2012) Aptamers and their biological applications. Sensors, 12(1):612–631.
  2. Sun H, Zhu X, et al. (2014) Oligonucleotide aptamers: New tools for targeted cancer therapy. Mol Ther Nucleic Acids, 3:e182.
  3. Brody EN, Gold L. (2000) Aptamers as therapeutic and diagnostic agents. J Biotechnol, 74:5–13.
  4. Murphy MB, Fuller ST, et al. (2003) An improved method for the in vitro evolution of aptamers and applications in protein detection and purification. Nucl Acids Res, 31(18):e110.
  5. Tombelli S, Minunni M, Mascini M. (2007) Aptamers-based assays for diagnostics, environmental and food analysis. Biomol Eng, 24(2):191–200.
  6. Ouellet E, Foley JH, et al. (2015) Hi‐Fi SELEX: A high‐fidelity digital‐PCR based therapeutic aptamer discovery platform. Biotechnol Bioeng, 112(8):1506-1522.
  7. MAWs; see http://2015.igem.org/Team:Heidelberg/Overview [Accessed 22 Feb, 2016]; and Prediger E. (2016) Functional nucleic acids as antibody alternatives for small molecule detection. [Online] Coralville, Integrated DNA Technologies. Available at http://www.idtdna.com/pages/decoded/decoded-articles/synthetic-biology/decoded/2016/02/04/functional-nucleic-acids-as-antibody-alternatives-and-for-small-molecule-detection. [Accessed 22 Feb, 2016].
  8. Proske D, Blank M, et al. (2005) Aptamers–basic research, drug development, and clinical applications. Appl Microbiol Biotechnol, 69(4):367–374.
  9. Keefe AD, Pai S, Ellington A. (2010) Aptamers as therapeutics. Nat Rev Drug Discov, 9(7):537–550.

Related reading

DNA oligonucleotide resuspension and storage—Core concepts: Get guidelines and recommendations for how to resuspend and store newly synthesized oligonucleotides.

Functional nucleic acids as antibody alternatives for small molecule detection—Research profile: Learn how 2015 iGEM Team Heidelberg students applied functional nucleic acids to design high-affinity, ligand-specific aptamers, in just hours, and without SELEX. They used their aptamers to replace conventional antibodies in western blots to successfully detect target proteins. Additional projects addressed repair of the mutated CFTR mRNA expressed by people afflicted with cystic fibrosis, and development of an in-the-field, date-rape, drug test strip. Their DNA aptamer sequence designs were synthesized as single-stranded DNA oligonucleotides by IDT.

Oligo synthesis: Why IDT leads the oligo industry—Core concepts: Read about the phosphoramidite method of oligonucleotide synthesis that IDT uses in its manufacturing processes. We also highlight the additional measures we take to ensure our customers receive the highest quality oligos and nucleic acid products in the shortest time possible.

Author: Ellen Prediger, PhD, is a senior scientific writer at IDT.

© 2016 Integrated DNA Technologies. All rights reserved. Trademarks contained herein are the property of Integrated DNA Technologies, Inc. or their respective owners. For specific trademark and licensing information, see www.idtdna.com/trademarks.


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