In The November Issue
• Next Gen Sequencing for Aptamer Selection
• Bioinformatics Service for Aptamer Selection
• Selecting Aptamers to Cells for Biomarker Discovery
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Next Gen Sequencing for Aptamer Selection
The introduction of next generation sequencing has enabled the analysis of millions of potential aptamer sequences in record time. In addition to a final enriched library, multiple rounds of SELEX can be analyzed to evaluate enrichment over time and identify high-affinity sequences. Final aptamer pools or pools from several selection rounds may be submitted for sequencing at Base Pair.
Bioinformatics for Aptamer Selection
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. We have proven techniques for choosing the right aptamer sequences.
Selecting Aptamers to Cells for Biomarker Discovery
Disease diagnosis and drug development rely on specific targets, or biomarkers, that discriminate between normal and diseased cells. Identifying early indicators of disease is a time-consuming process. (4). For diseases lacking diagnostic biomarkers and therapeutic targets, aptamers are providing new hope. Unlike antibodies, which are typically raised against a purified target protein, aptamers can be selected for binding to whole cells. By selecting for aptamers that bind to a specific type of cancer cell, but do not bind to normal cells, researchers are discovering aptamers that exclusively target diseased cells. While these aptamers can sometimes be used in diagnostics and as direct therapeutic or drug delivery/cell targeting agents, aptamer binding can be analyzed to reveal novel biomarkers. Biomarkers discovered through cell-SELEX can be used for production of more traditional diagnostic and therapeutic agents.
Biomarkers discovered through cell-SELEX can be used for production of more traditional diagnostic and therapeutic agents.
Selecting Aptamers to Cells
Cell-SELEX is an exciting technique for the creation of affinity agents without prior identification of a specific target. Researchers can isolate aptamers based on surface differences between normal and cancer cells and between different types of cancer cells (2,3). Researchers can even select for aptamers that are internalized into cells (4). Aptamers to multiple cell surface targets can be discovered in a single cell-SELEX process (1,3). Researchers seeking earlier diagnosis in brain glioma, gastric cancer, and breast cancer metastasis have recently turned to cell-SELEX.
Gliomas account for approximately 70% of brain cancer. Identification of biomarkers for early detection would accelerate treatment and improve the low survival rate. Researchers in China used the T98G glioblastoma multiforme cell line for positive aptamer selection and performed negative selection using the human fetal glia cell line SVGp12. After 9 cycles of cell-SELEX, they discovered two DNA aptamers with high affinity (1 – 3 nM) and selectivity for glioblastoma multiforme cells. The aptamers detected target in undiluted fetal bovine serum and cerebrospinal fluid. Decreased signal upon treatment with trypsin confirmed aptamer binding to extracellular targets. The synergistic activity of the two aptamers suggests recognition of two different binding sites or to different targets on the T98G cells (5). While the aptamer itself has likely utility for targeted drug delivery, identification of the aptamer target would further enable development of novel therapeutics and diagnostic tests.
Researchers at China Medical University and the Fujian Medical University used cell-SELEX to discover aptamers with high affinity and selectivity for metastatic circulating tumor cells (CTCs). The highly metastatic MDA-MB-231 cell line was used for positive selection and low-metastatic MCF-7 cells were used for negative selection. Following ten rounds of selection, aptamer M3 was chosen based on affinity and selectivity. The aptamer demonstrated high affinity for highly metastatic cell lines and no binding to normal cells. Using a streptavidin-coated plate and biotinylated aptamer, the aptamer successfully captured target cells spiked into non-target cells and whole blood. It was shown to function at both physiological temperature and room temperature and was stable in plasma over the required time frame (3). Aptamer M3 is a promising agent for enrichment and detection of highly metastatic CTCs.
Researchers at Lanzhou University in China used cell-CELEX to identify a biomarker for early stage gastric cancer. A mixed cell population from individual donors with early-stage gastric cancer was used for positive aptamer selection. Normal gastric mucosa was used for negative selection. After 12 rounds of cell-SELEX, aptamer Ap7 was selected based on affinity and selectivity. Binding of fluorescein-labeled aptamer was used to confirm selectivity for early stage gastric cancer cells. Further analysis was performed to characterize the discovered biomarker for early stage gastric cancer (6).
Characterizing Aptamer Targets
While aptamers to specific cells can be directly used in diagnostics, therapeutics and cell targeting, they can also be used to capture and identify novel aptamer targets. As early as 2008, researchers described a process for selecting aptamers to specific cells, then isolating and characterizing the aptamer targets. Following selection of aptamers, biotinylated aptamer and streptavidin-coated magnetic beads are used to isolate aptamer-bound cells. Soft cell lysis is performed to maintain aptamer-cell complexes and a magnet is used to separate aptamer-target complexes from cellular debris. The complexes are denatured and unknown targets are analyzed via mass spectrometry (1).
In the case of aptamer Ap7, further analysis revealed a promising biomarker for early stage gastric cancer. Based on MALDI-TOF mass spec and the NCBI database, the target protein was identified as peroxiredoxin-4. ELISA analysis confirmed higher expression of peroxiredoxin-4 in cells from early-stage gastric cancer than late-stage gastric cancer and normal gastric mucosa (6). Similarly, further characterization of aptamers to glioblastoma multiforme and circulating metastatic tumor cells may reveal new therapeutic targets and diagnostic biomarkers for early stage brain gliomas and recurring breast cancer.
Custom Aptamer Discovery Using Cell-SELEX
Cell-SELEX is an exciting technique that is currently underutilized in pre-clinical research. Researchers spend significant time and money testing commercially-available array products to identify biomarkers for critical diseases, often with ambiguous results. Selection of aptamer affinity reagents to cells (and unknown targets) of interest is a more direct way of identifying new disease-specific targets for therapeutic development and early-stage diagnostics.
Contact Base Pair today to learn more about aptamer selection to the specific cell line or cell population of interest to you.
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1. Berezovski, M.V., et al. Aptamer-facilitated biomarker discovery (Apta BiD). JACS Articles. 2008. 130:9137-9143.
2. Kaur, H., et al. Recent developments in cell-SELEX technology for aptamer selection. Biochimica et Biophysica Acta. 2018. 1862(10):2323-2329.
3. Li, W.M., et al. Selection of metastatic breast cancer cell-specific aptamers for the capture of CTCs with a metastatic phenotype by cell-SELEX. Molecular Therapy: Nucleic Acids. 2018. 12:707-717.
4. Pang, X., et al. Bioapplication of cell-SELEX-generated aptamers in cancer diagnostics, therapeutics, theranostics, and biomarker discovery: a comprehensive review. Cancers. 2018. 10(2):47
5. Wu, Q. DNA aptamer from whole cell-SELEX as new diagnostic agents against glioblastoma multiforme cells. 2018. 143:2267-2275.
6. Xu, P., et al. Peroxiredoxin-4 as a potential biomarker of early gastric cancer screened by cell-SELEX. Translational Cancer Research. 2017. 6(2)