Antibody-based diagnostic tests are widely used to detect, characterize, and monitor disease. For some diseases, development of specific antibody reagents and simple, antibody-based tests has proven particularly challenging. In many of these cases, diagnostic aptamers are enabling the development of tests that are faster, more robust, more user-friendly, and more accessible than ever before.

Infectious Disease

There is a widespread need for point-of-care tests that are faster, more sensitive, more user-friendly, and more stable. In many cases, the goal is to replace labor- and time-intensive cell culture and RT-PCR methods. In some cases, researchers are utilizing aptamers to generate plate-based colorimetric or fluorescent assays with improved sensitivity and specificity versus current antibody-based ELISA methods. (1) Aptamer-based lateral flow assays offer a simple, stable format for point-of-care and field-based use.

Pathogenic Bacteria, Bacterial Toxins, and Viruses

With the rapid spread of bacterial strains demonstrating multi-drug resistance, characterization of pathogens is critical.

Clostridium difficile, c. diff,  bacterium
c. diff

In the hospital setting, improved assays for rapid detection of bacterial infections including Clostridium difficile (C. diff) bacteria/toxin and methicillin-resistant Staphylococcus aureus (MRSA) would enable faster, more effective treatment. Because aptamers are easily selected for binding to toxic compounds and pathogens that would be harmful to antibody-producing organisms, aptamers are ideal reagents for infectious disease. Researchers at Jiangnun University developed a fluorescent bioassay using selective aptamers to Salmonella typhimurium. Rather than targeting a specific surface protein or toxin for selection, researchers used live bacteria. (2) Specific toxins or surface proteins, whole cells, and even live bacteria can be used to select aptamers. The PBP2a/MRSA-specific Base Pair aptamers show affinity for both the PBP-2a (penicillin-binding protein 2a) recombinant protein and MRSA bacteria.

 

Viral detection assays are generally very time-consuming. Field-based screening for tropical viruses, including avian influenza (H5N1, H1N1), dengue fever, West Nile virus, and Chikungunya are important for disease monitoring, treatment, and management of the spread of disease. (1) Base Pair is currently working with UCF and the Gates Foundation to develop aptamers for Zika, Chikungunya, and dengue virus using mosquito saliva. Because aptamers are stable in a wider range of temperatures than antibodies, aptamer-based ELISAs and LFAs are ideal for testing in tropical areas widely affected by mosquito-borne diseases.

Oncology

Early detection and accurate differentiation of cancer cells from normal cells are of utmost importance in cancer screening. The most aggressive cancers are often detected too late for successful treatment. Aptamers offer some unique abilities that are being applied to improve cancer detection.

Circulating and Tissue-Based Cancer Biomarkers and Cancer Cells

breast cancer tumor image
Breast cancer tumor

Selective aptamers for secreted biomarkers and circulating cancer cells in breast cancer, acute myeloid leukemia, small cell lung cancer, and other forms of cancer are being used to develop more specific blood-based assays with enhanced sensitivity. Similarly, selective aptamers are improving differentiation of tumor vs. normal tissue in MRI (magnetic resonance imaging), CT (computed tomography), and fluorescence imaging. (1) Pancreatic cancer is often diagnosed at an advanced stage where there is little chance of effective treatment. Based on findings related to the role of cancer stem cells (CSCs) in the pathogenesis of cancer, researchers at the Yonsei University College of Medicine in Seoul, South Korea developed aptamers selective for circulating pancreatic tumor cells exhibiting the characteristics of cancer stem cells. Aptamer binding was shown to be co-localized with CD133, a surface marker in HPAC (human pancreatic adenocarcinoma epithelial) cells. (3) Researchers in Australia have developed highly-selective RNA aptamers to the AC133 epitope of the cancer stem cell surface marker CD133. The researchers showed improved tumor penetration and retention when compared with a CD133-specific antibody. (4) Cancer cell-specific aptamers, with enhanced selectivity and tissue penetration, reduced immunogenicity, and a multitude of detection options, offer exciting opportunities in blood-based cancer screening, molecular imaging and targeted therapeutics.

Structure-Switching Aptamers

Aptamers can detect small differences between very similar molecules. They can be used to differentiate between similar bacterial strains and between cancerous and non-cancerous cells. Aptamers can be conjugated to a wide range of detection molecules without loss of affinity or selectivity, enabling enhanced in vitro and in vivo imaging. Perhaps the most compelling feature of aptamers in diagnostics is their ability to change conformation upon binding. Structure-switching aptamers can be designed to generate signal (fluorescent, electrochemical, etc.) upon binding to their target. This self-signaling ability enables fast, selective detection in complex samples without wash steps. Structure-switching aptamers are being utilized in the development of selective biosensors and simple, rapid detection assays in a growing number of fields. Aptamer-based biosensors, or “aptasensors”, have been used for detection of circulating cancer biomarkers including VEGF165, direct detection of cancer cells, and even a chemiluminescent imaging array. (5) Base Pair has developed structure-switching aptamers for several compounds, including drugs of abuse and the stress hormone cortisol, and has several on-going projects involving structure-switching aptamers.

Contact Base Pair to learn more about structure-switching aptamers and aptamer development for biosensors, ELISA-like assays, LFAs, imaging, and other applications.

References:

C. diff image courtesy of CDC Breast cancer tumor image courtesy of Joseph Szulczewski, David Inman, Kevin Elicein, and Patricia Keely. National Cancer Institute, Carbone Center at the University of Wisconsin.

1.   Chandola, C. et al. Application of aptamers in diagnostics, drug-delivery and imaging. J. Biosci. 2016. DOI: 10.1007/s12038-016-9632-y.

2.   Duan, N. et al. Selection and characterization of aptamers against Salmonella typhimurium using whole-bacterium Systemic Evolution of Ligands by Exponential Enrichment (SELEX). J. Agric. Food Chem. 61:3229-3234. (2013). dx.doi.org/10.1021/jf400767d.

3.   Kim, Y.J., Lee, H. S., Jung, D. E., Kim, J. M., and Song, S. Y. (2016), The DNA aptamer binds stemness‐enriched cancer cells in pancreatic cancer. J Mol. Recognit. doi: 10.1002/jmr.2591

4.   Shigdar, S. et al. RNA aptamers targeting cancer stem cell marker CD133. Cancer Letters. 330:84-95. http://dx.doi.org/10.1016/j.canlet.2012.11.032

5.   Wu, Xu, et al. Aptamers: Active targeting ligands for cancer diagnosis and therapy. Theranostics. 2015. 5(4) :322-344.