From Molecular Diagnostics to Therapeutics with Aptamers

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One of the most demanding and challenging tasks of bioanalytical chemistry and bioseparation is the replacement of animal- and hybridoma-made antibodies that have high production cost and short shelf life with artificial competitors that can be easily selected and chemically synthesized in high purity at low cost. Aptamers as "synthetic antibodies" have enormous potential. These small single-stranded DNA or RNA oligonucleotides can bind with high affinity and specificity to a variety of different targets ranging from small molecules to proteins. They are affinity ligands with significant potential to change the field of affinity probes and to replace antibodies as diagnostic and separation applications. I propose the development of three related research themes: 1) affinity purification of cells by switchable aptamers, 2) detection and imaging of rare cells by aptamers, and 3) discovery of rare cell biomarker by aptamers and mass spectrometry.***Current antibody-based approaches for cell isolation and purification are not optimal because of the antibody's effect on cell viability and purity. In the first research theme, we will develop two types of DNA aptamers that bind specifically with high affinity to immune receptors and cancer stem cell receptors. The first type of aptamers will be EDTA-switchable DNA aptamers that bind to cells in the presence of Ca(II) or Mg(II) ions, and release the pure and intact cells upon addition of EDTA or EGTA in solution. The second type of aptamers will be "cold" switchable DNA aptamers that selectively bind to receptor positive cells at room temperature and release them at 0°C to yield aptamer-free cells.***In the second theme, DNA aptamers selected for lung adenocarcinoma cells from postoperative tissues will be used for sensing and counting rare cancer cells (<100 cells/ml) in blood. The enumeration and molecular profiling of rare cells, such as cancer circulating tumour cells (CTCs) and cancer stem cell (CSCs) in biological fluids, is an emergent methodology for the detection and treatment of cancer. Such tumour specific aptamers can be produced for individual patients and synthesized many times during anticancer therapy, thereby opening up the possibility of personalized diagnostics. ***The third theme will use the findings of the first two technologies such as aptamers to rare cells, and a cell isolation procedure for identification of new protein biomarkers by mass spectrometry. The important feature of this theme is that it yields synthetic affinity probes (aptamers) simultaneously with biomarker discovery. ***The combined activities of this program over the next 5 years will make a significant contribution to the development of new in vitro evolution techniques for nucleic acid aptamers, and the design of new bioassays and biosensors.**