In This Issue:
- Vote Today for the Upcoming Webinar Topic and Enter to Win a $50 Visa Gift Card
- Aptamers for Biomarker Discovery and Detection
- New Aptamer to Amino Acid Citrulline: Biomarker for Renal and Intestinal Failure
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Aptamers for Biomarker Discovery and Detection
Aptamers for Biomarker Detection
Biomarkers are generally proteins or molecules that can be correlated with disease. In some instances a protein or molecule may be present or missing. In others, it may be upregulated or downregulated. Because a single protein or molecule may be associated with a number of diseases involving similar pathways, a panel of biomarkers, or biomarker profile, is often used to detect, monitor, and manage treatment for a wide range of conditions. Many biomarker assays are based on the use of antibodies. While these assays offer a great deal of specificity, sensitivity, and convenience, there are some limitations. For starters, developing antibodies to non-immunogenic molecules or infectious agents (small molecules, metabolites, pathogenic bacteria, virus-infected cells, toxins) is difficult, as antibody generation involves living immune cells. Antibodies are often unable to distinguish between highly similar molecules and heterophilic antibodies and anti-animal antibodies can interfere with antibody-based assays. Antibodies also lack high temperature stability, impairing the use of antibody-based assays in tropical climates. Unlike antibodies, aptamers can be selected against toxins and small molecules. Aptamers can be selected to distinguish between highly similar molecules and they are stable at elevated temperatures. Once an aptamer is selected it can be chemically synthesized, a process that is highly reproducible and desirable for long-term biomarker assay production. Aptamer use is being explored in a wide range of biomarker assays, including LFAs, sensors, and ELISA-like assays. (1)
Researchers in Singapore used an aptamer-nanosheet sensing platform to detect Plasmodium lactose dehydrogenase protein (pLDH), a biomarker for malaria. Traditional malaria tests involved cell staining and optical microscopy, requiring specialized equipment and technicians. This method was time-consuming and lacked sensitivity, resulting in some false negatives. New rapid detection tests (RDTs), often antibody-based, are sensitive to the high temperatures in many tropical areas. In the aptamer-based sensing platform, an aptamer selective for pLDH was fluorescently-labeled and combined with MoS2 (molybdenum disulfide) nanosheets. The sheets bind nucleic acids and quench fluorescence. Concentrations were optimized to ensure aptamer binding and quenching. Upon addition of the target protein, pLDH, the aptamer released from the nanosheets and preferentially bound to the target protein, restoring fluorescence. Detection of pLDH levels down to 550 pm was achieved in 10 minutes and met clinical requirements for the malaria biomarker. (3)
Researchers in South Korea developed an aptamer-based sandwich assay for the detection of Lipocalin-2, a biomarker for hepatocellular carcinoma (HCC). Researchers first selected aptamers to Lipocalin-2, then screened the aptamers via dot blot for identification of a matched pair. The capture aptamer was labeled with a 5’ amine and bound to a DNA binding plate. The detection aptamer was labeled with HRP. The resulting assay was able to detect lipocalin-2 in the linear range of 2.5 to 500 ng/mL in undiluted serum. Similar assays could be developed for biomarkers present in the ng/mL and ug/mL range, including Serum Amyloid A (SAA) and C-reactive Protein (CRP). The ability to generate stable aptamer-based assays that require no sample processing or dilution can be applied to a wide range of field-based, point-of-care biomarker tests for diagnosis, disease monitoring, and treatment monitoring. (4)
Aptamers in Biomarker Discovery
During the traditional aptamer selection and enrichment process, known as SELEX, a large library of DNA or RNA oligonucleotides is exposed to a target protein. Following many rounds of binding, elution, and amplification, a handful of aptamers with desirable characteristics and binding properties are selected. Whole cell SELEX, or Cell-SELEX, can be utilized to select aptamers that bind specific cell types, including cancer cells or virus-infected cells. In Cell-SELEX, the target protein or molecule is replaced by live cells and the aptamers, rather than the target cells, are often immobilized. Normal cells are often used for several rounds of negative selection to improve selectivity for infected or cancerous cells. Once a new aptamer is selected, it’s binding partner can be isolated and analyzed. Cell-SELEX enables cell type-specific biomarker discovery. (2)
As early as 2008, researchers were reporting successful biomarker discovery via whole cell SELEX coupled with mass spectrometry. Researchers at the University of Florida generated aptamers selective for whole leukemia cells. One aptamer was selective for T-All (T-cell acute lymphoblastic leukemia), AML (acute myeloid leukemia), and some B-ALL (B-cell acute lymphoblastic leukemia) cells. Cells were lysed and membrane proteins were solubilized. Biotinylated aptamer and SA-coated magnetic beads were used to isolate the aptamer-target protein complexes. Captured proteins were separated via SDS-Page. Protein bands were analyzed via LC-MS/MS. The target protein was identified as protein tyrosine kinase-7, or PTK7, a transmembrane protein. (5) Several other groups have developed aptamers to specific cell types. In many cases, aptamers bind known cell surface markers, but aptamer selection has led to the discovery of novel biomarkers as well.
The unique process of aptamer selection offers exciting opportunities for biomarker discovery, while aptamer selectivity and stability make them ideal reagents for biomarker assay development.
Please CONTACT US for information about development of aptamers to traditional protein biomarkers and small molecule biomarkers and cell-based aptamer selection for biomarker discovery.
1. Jayasena, S. D. Aptamers: An emerging class of molecules that rival antibodies in diagnostics. Clinical Chemistry. 1999. 45(9):1628-1650. (1)
2. Jin, C. et al., Cancer biomarker discovery using DNA aptamers. Analyst. 2015. 10.1039/C5AN01918D.
3. Kenry, et al. Highly sensitive and selective aptamer-based fluorescence detection of a malarial biomarker using single-layer MoS2 nanosheets. ACS Sens. 2016. 1 (11), pp 1315–1321 doi: 10.1021/acssensors.6b00449
4. Lee, K. et al. Aptamer-based sandwich assay and its clinical outlooks for detecting lipocalin-2 in hepatocellular carcinoma (HCC). Scientific Reports 2015. 5, Article number: 10897. doi:10.1038/srep10897
5. Shangguan, D. et al. Cell-specific aptamer probes for membrane protein elucidation in cancer cells. Journal of Proteome Research. 2008. 7(5):2133-2139. Doi:10.1021/pr700894d
Aptamer to Amino Acid Citrulline: Nutritional Supplement and Biomarker for Renal and Intestinal Failure
Citrulline (CIT) is an amino acid involved in arginine (ARG) metabolism. It is named after the watermelon, Citrullis Vulgaris, from which it was first isolated. Citrulline binds the hydroxyl radical, protecting DNA and metabolic enzymes from oxidation. It is involved in nitrogen balance and muscle homeostasis. Circulating citrulline is a biomarker for renal and intestinal failure. Because citrulline is metabolized in the kidneys, elevated levels of citrulline are an indication of renal failure. Conversely, citrulline is produced in the small intestine. Low level of plasma citrulline is an indication of poor intestinal absorption and intestinal failure. Citrulline has been shown to stimulate muscle protein synthesis and has become a popular nutritional supplement (7). Studies have shown that L-citrulline decreases muscle soreness post-workout. (6)
For non-immunogenic small molecules like the amino acid citrulline, aptamer selection is often chosen over antibody production. Base Pair citrulline aptamer ATW0100 was recently used successfully in a biosensor development project. The article “SERS competitive binding biosensor development utilizing surface modification of silver nanocubes and a novel citrulline aptamer” is currently in press at the Journal of Biomedical Optics. The binding curve for the citrulline aptamer using microscale thermophoresis is shown below.
6. Erika Gebel. Watermelon juice prevents aching muscles. Chemical & Engineering News. 2013. http://cen.acs.org/articles/91/web/2013/07/Watermelon-Juice-Prevents-Aching-Muscles.html
Accessed July 19, 2017.
7. Christophe Moinard and Luc Cynobar. Citrulline: A new player in the control of nitrogen homeostasis. J. Nutr. 2007. 137:1621S-1625S.