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Enzyme-linked DNA aptamer assay provides advantages over standard immunoassays

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.

Citation summary: Find out how this research team used candidate aptamers in an enzyme-linked aptamer sorbent assay (ELASA) to detect human insulin-like growth factor-I, a biomarker for recombinant human growth hormone, used in athletic doping.

Mar 28, 2016

Revised/updated Feb 15, 2017

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

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.

IDT synthesizes aptamers and aptamer libraries

IDT-synthesized aptamers and aptamer libraries have contributed to hundreds of published research papers which describe the successful use of such sequences (see the Related Citations sidebar).

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.

Base modifications increase aptamer options

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.*

How to order aptamers

To order aptamers online, visit the Custom DNA Oligos or Custom RNA Oligos ordering pages. Enter your desired scale, purification, and the sequence(s) with random bases or modifications to suit your needs.

*For aptamer sequence designs with greater than 20 2′-Fluoro bases inserted, or to include 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 Care.

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