Grant: Research Tools and Instruments - Category 1 (<$150,000)

The proposed instrument, a flow cytometer, is pivotal to our research on aptamer selection for targets on the cell surface and inside cells. Aptamers are short synthetic DNA (or RNA) oligonucleotides selected from large libraries of random DNA sequences for the ability to bind target molecules by a process termed SELEX (systematic evolution of ligands by exponential enrichment). Similar to antibodies, aptamers scan serve as: (i) affinity probes in quantitative immunoassays, (ii) contrast probes in whole-body imaging, (iii) drugs, and (iv) "vehicles" for drug delivery. Apatamers have so far been selected for significantly fewer targets and utilized in significantly fewer applications than antibodies. One of the reasons for this is the lack of efficient methods for aptamer selection. Such methods have been a research focus at my laboratory for the last 5 years. In the first stage of our research, we advanced methods for selection of pure-target aptamers by pioneering selection of "smart" aptamers (JACS 2005, 127, 3165; JACS 2005, 127, 11224) and non-SELEX selection of regular aptamers (JACS 2006, 128, 1410; Nature Protocols 2006, 1, 1359). Smart aptamers have made possible quantitative immunoassays with an ultra-wide dynamic range of target concentrations (JACS 2007, 129, 7260). Our current focus is the development of aptamers for targets on the cell surface and inside cells, as well as the use of such aptamers for studies in live cells and whole organisms. In our preliminary research, we demonstrated selection of highly-potent aptamer pools for targets on the surface of live cells (JACS 2008, 130, 9137) and in crude cell lysate (Nucleic Acids Res., 2009, 37, e62); we are now working on a method for selection of aptamers with targets inside cells. These methods create the foundation for highly-innovative aptamer-based biomedical technologies for: (i) the study of single cells, (ii) biomarker discovery, (iii) cell sorting, (iv) diagnosis of metastases, etc. The development of aptamers for cellular targets and their further use in the above five directions is impossible without a flow cytometer. This application requests funds for purchasing a flow cytometer to support our research on aptamers for cellular targets.