In The December Issue
• Aptamer Maturation: Improving Aptamer Performance
• NGS and Bioinformatics Services for Aptamer Selection
• Aptamers in Flow Cytometry
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Aptamer Maturation: Improving Aptamer Performance
A key advantage of aptamers is the ability to select aptamers in vitro for binding to difficult (non-immunogenic, toxic) targets. A typical selection is completed in less than eight weeks. But what if selected aptamers do not achieve the degree of affinity or stability required for a specific end-use application? All is not lost. There are a number of aptamer modifications that can be performed post-selection to enhance aptamer performance. If you haven’t yet seen our latest article on aptamer maturation, click the button below to learn more.
Next Gen Sequencing and Bioinformatics for Aptamer Selection
The introduction of next generation sequencing has enabled the analysis of millions of potential aptamer sequences in record time.Choosing the right aptamer sequences for further testing is a critical step in the biomarker discovery process. Whether you are new to aptamer discovery or simply short on manpower, Base Pair can help. Learn more about NGS and Bioinformatics Services from Base Pair.
Aptamers in Flow Cytometry
Flow Cytometry enables analysis of cell populations at the single cell level. During analysis, single cells pass through a laser in a fluid stream. Light scattering and fluorescence signal are used to identify important cell characteristics and the presence of cell surface markers of interest. Flow cytometry is often used to differentiate between normal and cancer cells and types of tumor cells in clinical diagnostics and oncology research. It is used to perform cell phenotyping in stem cell research and to identify specific pathogens in microbiological analysis and environmental testing (4).
Advantages of Aptamers in Flow Cytometry
Aptamers are small strands of DNA or RNA that are selected to bind a specific target of interest (Learn more about aptamers and aptamer selection.) Aptamers can be selected to a variety of different targets in vitro, including proteins, small molecules, viruses, bacterial pathogens, and toxins. Chemical synthesis can make aptamers more reproducible and cost-effective than other affinity reagents. Aptamers can also be conjugated to a wide range of fluorophores and functional groups without any loss of selectivity or affinity. Aptamers are being conjugated to a growing variety of nanoparticles for more sensitive detection in flow cytometry (3,4).
Using a technique called Cell-SELEX, aptamers can be selected to bind a specific cell type without prior knowledge of a specific target on the cell surface. (Learn more about Cell-SELEX.) Because targets in Cell-SELEX are in their natural conformation on the cell surface, aptamers generated can be highly effective in flow cytometry (1,4).
Selection of Aptamers for Flow Cytometry using Cell-SELEX
Researchers in New Zealand used cell-SELEX to identify aptamers selective for live Pseudomonas aeruginosa grown in biofilm. Flow cytometry was used to assess binding of aptamer libraries during the cell-SELEX process and to evaluate binding of the final aptamer candidates. Combining flow cytometry and cell-SELEX, researchers were able to select an aptamer that discriminates P. aeruginosa from other gram-negative bacteria without prior identification of a specific target or epitope (5).
Researchers in Germany used cell-SELEX to discover aptamers selective for CD24+ HT-29 cells. Flow cytometry was used to evaluate aptamer binding and select the aptamer with the greatest affinity and selectivity. Fluorescence generated with the anti-CD24 aptamer and positive cells was almost 9 times greater than background and was 33% higher than the signal-to-noise observed for the CD24-specific antibody used for comparison. This aptamer has potential in both diagnostic and therapeutic applications (2).
Selection of Specific- and Pan-Cell Binders for Flow Cytometry
While selectivity for a certain strain of virus or bacteria is often important, the ability to detect a variety of strains through the use of aptamer cocktails or pan-binders is sometimes beneficial as well. Binding to multiple cell surface targets can enable detection of strains with slight differences in surface expression and detection of bacteria and viruses which undergo small changes intended to avoid detection (4). Base Pair’s patented multiplex SELEX technology can be used to select aptamers to multiple targets in a single selection and isolate specific and pan-binders. (Learn more about Base Pair’s VENNmultiplexTM SELEX.) Multi-parameter flow cytometry can be used to evaluate aptamer binding to several strains or cell types in a single experiment.
Custom Aptamer Selection
There are many options to consider when selecting aptamers for use in flow cytometry. Aptamers can be selected through traditional SELEX using recombinant proteins or extracellular peptides, then screened using flow cytometry. Aptamers can also be discovered through cell-SELEX using flow cytometry to evaluate the final aptamer clones. In some cases, a hybrid approach using some rounds with a purified target and some rounds with positive and negative cell lines may work best. Base Pair will develop a selection strategy to best meet your specific project requirements.
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1. Berezovski, M.V., et al. Aptamer-facilitated biomarker discovery (Apta BiD). JACS Articles. 2008. 130:9137-9143.
2. Fafinska, J. et al. DNA aptamers for the malignant transformation marker CD24. Nucleic Acid Therapeutics. 2018. 28(6).
3. Jayasena, S. D. Aptamers: An emerging class of molecules that rival antibodies in diagnostics. Clinical Chemistry. 1999. 45(9):1628-50.
4. Meyer, M., et al. Aptamers: versatile probes for flow cytometry. Applied Microbiology and Biochemistry. 2013. 97(16):7097-7109.
5. Saundry, Jennifer and Darren Day. Selection of DNA aptamers specific for live Pseudomonas aeruginosa. PLoS ONE. 12(9):e0185385.