A simple 384-well assay to assess cellular response to aptamer exposure
Aptamers are a special class of single-stranded nucleic acid (DNA) molecules. Since their discovery in the early 1990s, significant effort has been made to make aptamers clinically relevant for many diseases, including cancer. With advances in targeted therapy and imaging, aptamers are now considered as potential targeting ligands because of their very high specificity and affinity to bind surface tumor markers, as well as for their reproducible chemical synthesis. They are characterized by their small size and ease of modification for conjugation with radioactive moieties, tags, and fluorophores.
Aptamers can be used in numerous biomedical applications, from diagnostics and patient stratification to cancer progression and even therapy. The first aptamer-based therapeutic was approved by the FDA in 2004.
By using sequential DNA library enrichments with MCF7 cells, PCR, and two different DNA sequencing techniques, Laia Civit identified four aptamers that show interesting behavior on breast cancer cells and on a set of other cancer cell lines (A549, Ramos and THP1). To measure the potential innate response through the Toll-like receptors, the four aptamers were further tested in a 384-well cell-based assay with immortalized murine embryonic stem cell-derived macrophages. Compared to positive controls, negligible immunogenicity was confirmed by quantifying the release of TNF from those cells exposed to aptamers for 24 hours. All four were confirmed to be non-immunogenic, as expected.
Many aptamers are now in varying stages of development, from pre-clinical studies to clinical trials, and some are even in the FDA process for approved treatments. These aptamers open new horizons as highly potent drugs or diagnostic reagents. Like chemical compounds or therapeutic antibodies, they need to be screened in an easy manner that is compatible with HTS. Read this article to find out more and learn how to do it!
The sensitive and specific detection of pathogenic cells is essential in clinical diagnostics. To achieve this, molecular tools are required that unequivocally recognize appropriate cell surface molecules, such as biomarkers that come along with disease onset and progression. Aptamers are short single-stranded oligonucleotides that interact with cognate target molecules with high affinity and specificity. Within the last years they have gained an increased attention as cell-recognition tools. Here, we report a systematic analysis of a cell-SELEX procedure, for the identification of aptamers that recognize breast cancer cells. Besides a comparison of conventional (Sanger) with high-throughput sequencing techniques (next-generation sequencing), three different screening techniques have been applied to characterize the binding properties of selected aptamer candidates. This method has been found to be beneficial in finding DNA aptamers, rarely enriched in the libraries. Finally, four DNA aptamers were identified that exhibit broad-spectrum interaction patterns to different cancer cell lines derived from solid tumors.
: Biochimie, 2018 Feb; 145:53-62.