Small RNAs/Functional Genomics
Support and Educational Content

Enzyme-linked DNA aptamer assay provides advantages over standard immunoassays

IDT 5′-biotinylated aptamer library with randomized bases leads to rhGH aptamer candidate

Bruno JG, Richarte AM. (2016) 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.

Background

Insulin-like growth factor-I (IGF-I; aka somatomedin C) can be used as a biomarker for detection of athletes doping with recombinant human growth hormone (rhGH). This research group describes the development of a simple, aptamer-magnetic bead (MB)-based assay for serum human IGF-I. The IGF-I aptamer-MB assay could offer a significant improvement over immunoassays, which currently require purification of IGF-I from other binding proteins and deliver inconsistent performance results. Use of DNA aptamers in place of antibodies provides a binding reagent of a defined composition that can be manufactured with high fidelity. Aptamers also display superior target affinity, when appropriately selected. In addition, they can be produced more quickly and cost-effectively, as they require no animal host.

Experiment

The researchers identified 72-base aptamer candidates using 9 rounds of MB-SELEX DNA aptamer development. 36 aptamer candidates, supplied by IDT as 5′-biotinylated DNA oligonucleotides, were tested for affinity against microplate-bound, recombinant human IGF-I (rhIGF-I). The top 4 aptamer candidates were tested in both capture aptamer-MB and reporter aptamer roles, and were compared in an enzyme-linked aptamer-MB sandwich format (ELASA, enyzme-linked aptamer sorbent assay) in undiluted human serum.

Results and conclusion

The assay was reproducibly sensitive with a probable limit of detection <16 ng/mL in human serum and did not require the separation if rhIGF-I from IGF binding proteins (IGFBPs). Based on these results, the authors suggest that the aptamer with superior binding, binds to an exposed epitope in rhIGF-I:IGFBP complexes.

In ongoing experiments, the authors will assay actual isolated rhIGF-I:IGFBP complexes in buffer but here conclude that the assay works to some extent in human serum, especially when excess rhIGF-I is present.

Products from IDT

All of the DNA templates, primers, and the library of biotinylated candidate aptamers used in these experiments were synthesized by IDT. The 72-base DNA aptamers were designed with 18 fixed-sequence bases at each end to serve as primer binding regions for PCR amplification. The intervening 36 bases were randomized in the initial SELEX DNA template library.

Product focus

Aptamers

IDT synthesizes 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).

Most aptamers are 20–80 nt, single-stranded DNA or RNA sequences. However, IDT can synthesize longer aptamers, if needed. 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). You must provide your sequence designs, including specifying modifications and positions, for synthesis.

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:

Here is a selection of additional citations that use aptamers manufactured by IDT:

  • 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 biotin for purification.
  • 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.

Additional reading:

Planning to work with aptamers?—Learn about aptamers, SELEX, and how IDT can assist you with reagents for your aptamer applications.

IDT publications—Aptamers—IDT scientists also collaborate with outside research groups on aptamer design and aptamer applications. This link provides a list of those publications.

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.


siRNAs with increased potency

Use IDT DsiRNAs to engage the Dicer-processing/RISC-loading mechanisms and get increased silencing potency over traditional siRNAs.

Learn more ≫


Related Articles

RNAi and DsiRNA: Pathway, Mechanism, and Design

An overview of the RNA interference (RNAi) pathway, its applications, and design considerations for gene silencing experiments.

Read more ≫

Using Antisense Technologies to Modulate Noncoding RNA Function

Useful modifications and design considerations for effective antisense oligonucleotides.

Read more ≫

Small RNA Therapies for Cystic Fibrosis

DsiRNAs are used to develop a protocol for efficient small RNA delivery into airway epithelia to study of miRNA regulation of airway fluid and electrolyte transport.

Read more ≫

Using DsiRNA to Map Pain Pathways in the CNS

Canadian laboratory targets pain receptors for expression knock out via in vivo delivery of DsiRNA 27mer duplex RNAs into the mouse CNS.

Read more ≫

The Future of RNAi and Aptamer Technologies—An Interview with John Rossi

An interview with Dr John Rossi, whose research focuses on the biology and applications of eukaryotic small RNAs and, in particular, their therapeutic use in HIV/AIDS and cancer.

Read more ≫