The need to understand cancer biology at a cellular level requires a comprehensive interrogation of mRNA and proteins at high throughput and preferably simultaneously. The overall goal of the proposed research is to expand preliminary experiments by the applicant to further develop, validate and optimize magnetic nanosensors (magnetic relaxation switches, MRS) capable of sensing different types of molecular targets. In preliminary experiments we have shown that when monodisperse magnetic nanoparticles self-assemble into oligomeric nanoassemblies, there is a corresponding decrease in the spin-spin relaxation times (T2) of surrounding water detectable by NMR techniques. This fundamental observation has allowed us to measure DNA-DNA, protein-protein, small molecule protein interactions as well as enzyme activities (Nat Biotech 2002;20, 816-820). The magnetic relaxation switch (MRS) technique is exceedingly sensitive, allowing the detection of target molecules at femtomole levels with bench-top detectors and attomole levels by high-resolution MR imaging. The assay is performed in solution and does not require isolation or purification of the sample. In the proposed research the applicant will pursue three specific aims using telomerase as a cancer related target: (1) design and optimize MRS probes that independently recognize telomerase-mRNA, -protein and enzyme activity; (2) develop and optimize magnetic resonance techniques for simultaneous screening of telomerase-mRNA, -protein and -activity; and (3) develop and optimize internalization protocols for telomerase MRS probes that would allow detection of a target inside intact cells. It is hypothesized that telomerase mRNA, telomerase protein and telomerase activity can be detected in cell lysates and tissue extracts without prior purification or amplification using the developed MRS technique. Major biotechnical and medical applications of the developed technique lie in 1) the development of techniques to simultaneously interrogate RNA and proteins, 2) the development of high-throughput solution phase arrays, and 3) the ability to image molecular interactions by magnetic resonance imaging. The proposed research will be conducted in a multidisciplinary environment and the integrated training and research program will enable the candidate to establish himself as an independent cancer researcher. ? ?
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