Celebrating DNA aptamers on DNA day
April 25th is DNA day. This day celebrates the completion of the Human Genome Project in 2003 and the discovery of DNA’s double helix in 1953.
DNA aptamers are cheaper and simpler to manufacture and more robust than their RNA counterparts. Because of this, DNA-based aptamers are often used as research reagents and affinity tools within diagnostics.
At Aptamer Group, we use DNA and RNA aptamer libraries to deliver the best Optimer for your specific project, so we are pleased to celebrate DNA day and the contribution that DNA-based aptamers have made to science and diagnostics.
Taking the heat out of hot-starts
Taq DNA polymerase used in PCR reactions can add bases to ssDNA in a non-template-dependent manner. At the high temperatures used during PCR thermal cycling, this activity is reduced, and annealing becomes more stringent. Yet, at lower temperatures, including room temperature, this activity can impact the reaction specificity, leading to erroneous results. Preventing the low-temperature activity and retaining the reaction specificity is crucial to the experiment.
‘Hot-start’ PCR offered a way to overcome the specificity issues. Approaches include excluding the Taq DNA polymerase enzyme and spiking it into the mixture once thermal cycling begins at high temperatures or using antibodies specific to Taq that could block the enzyme’s activity at room temperature. In the first approach, process issues include the labor-intensive requirements of opening all the tubes during the reaction and adding small volumes of reagents, risking contamination. Antibodies are still used in ‘hot start’ reactions, but NEB, the life science reagent supplier now offers a best in class alternative, that is based on DNA aptamers.
In their product, NEB use a SOMAmer, a specific type of aptamer from SomaLogic that includes specific nucleobase modification to improve the inhibition profile and reduce unintended side effects. Just as the antibody-Taq inhibitory solution worked, the Taq aptamer used by NEB inhibits Taq polymerase at room temperature. This DNA aptamer solution has proven to yield highly specific PCR reactions with additional advantages over the antibody-Taq inhibitor:
- The aptamer inhibition/activation process is fully reversible, preventing any further Taq activity at the end of thermal cycling.
- The aptamer inhibition is released at lower temperatures (~45°C) than the antibody (94°C), removing the high temperature activation step and enabling faster protocols.
Finding fungus in foods
Neoventures Biotechnology Inc. has developed OTA-Sense and AflaSense for the detection of mycotoxins in agricultural products. Both of these commercially available diagnostic assays use DNA aptamer-based sensing technology and offer highly sensitive detection of Ochratoxin A (OTA) from Aspergillus and Penicillium species for OTA-Sense, and aflatoxins from Aspergillus for AflaSense. These mycotoxins are known carcinogens, and the regulatory bodies have established maximum allowed limits within foodstuffs such as cereal, wine and baby food.
These diagnostic kits are fluorescent-based high throughput assays, consisting of two components: an aptamer-based clean-up column and a detection solution. Mycotoxins are extracted from the foodstuffs using an organic solvent, like acetonitrile, and then filtered through glass wool and loaded onto the mycotoxin-specific affinity column, where the OTA or aflatoxin specific aptamer is immobilized on a resin. After washing, the mycotoxin is eluted from the column and mixed with a detection solution containing mycotoxin-sepcific aptamers and terbium. The terbium acts like a bridge between the aptamer and mycotoxin, enhancing the fluorescence of the terbium several-fold. This fluorescence can be quantified as a read-out for the concentration of mycotoxin present in the food sample.
Ultrasensitive detection of NSCLC
A panel of protein biomarkers that correlate to non-small cell lung cancer are analyzed as part of Aptamer Sciences’ in vitro diagnostic AptoDetect-Lung. Four of the biomarkers are involved in cancer growth (EGFR1, MMP7, CA6 and KIT) and three are immune-related (CRP, C9 and SERPINA3). Using an aptamer-based bead microarray with DNA aptamers to each of the biomarkers, AptoDetect-Lung analyses the differential expression levels across the panel. This assay allows diagnosticians to distinguish between lung cancer and benign nodules in indeterminate pulmonary nodules with a specificity of 91.7% and sensitivity of 75%.
Keeping pace with a pandemic
As the COVID-19 pandemic emerged, Aptamer Group developed DNA aptamers specific to SARS-CoV-2 to enable rapid diagnostics and research around this new disease. Specific aptamers to the viral spike protein and nucleocapsid protein were generated in just 17 days. They are being progressed to commercial diagnostic products with partners to offer rapid SARS-CoV-2 antigen tests that can help to open economies and reduce the spread of the virus.
Flowing through cell separation
DNA aptamer reagents for cell separation by flow cytometry are available from Aptamer Sciences. Using aptamers to a range of biomarkers, including CD-31, EGFR and HER2, they have developed several magnetic bead-based cell isolation kits to efficiently isolate biomarker-positive cells. Enriched cell populations can be easily assessed using flow cytometry via the dual-labeled aptamers in these kits, with a biotin tag on one end of the aptamer for simple streptavidin capture on beads and a fluorescent tag on the other end for monitoring via flow cytometry. These kits offer a simple strategy to isolate biomarker-positive cells with high yield, purity and cell viability.
From deciphering the structure of double-stranded DNA to cracking the human genome, over the past nearly 70 years our understanding of DNA has advanced hugely. We are now engineering DNA molecules to offer better research reagents and improved and novel diagnostics using aptamer technology.