The overall goal of this proposal is to develop the ability to detect and quantitate changes in gene expression in real-time and with a degree of sensitivity suitable to monitor minor changes at a single cell level. This will have considerable value in basic biomedical research, disease mechanism studies and diagnosis, and drug discovery. This revised proposal centers on the use of molecular beacons (MBs) for the analysis of gene expression in living cells. In particular, it focuses on the further development of MBs for studying breast cancer cells and single neurons. During our previous research in studying mRNA expression in single cells, we have encountered several limitations of MBs for cellular analysis. These included susceptibility to degradation by nucleases and single strand binding proteins, incomplete quenching of the fluorophore, and weak fluorescence intensity of conventional fluorophores. The autofluorescence of the cells also proved to limit the sensitivity of MBs for the detection of genes with a low expression. To overcome these limitations, we have proposed many novel solutions including superquenching moieties, bright amplifying fluorescent polymers, and molecular beacons comprised of locked nucleic acid bases. While these advances have not been tested in vivo they have shown promise in solution experiments. In addition to improving MBs as effective molecular probes for intracellular monitoring, we will also integrate advanced imaging methods into our analysis. In our previous results, we have demonstrated the advantages of confocal imaging when applied towards intracellular analysis. In this proposal we plan to demonstrate the advantages of multiple photon imaging and fluorescence time resolved imaging for more sensitive detection of MBs by removing the biological background. Once the MBs are improved and the new techniques are developed we will focus on intracellular applications of MBs for the detection of mRNA using these advancements. Currently, several genes, MnSOD, Cyclin D1 and survivin, have been identified as cancer markers and have been studied in cancer genomics. Using MBs, we will focus on elucidating the effects that different anti-cancer agents have on these genes in different human breast carcinoma cell lines. Given the genetic foundation of learning and memory, we will also use MBs to explore the effects of extracellular stimuli on the gene expression in neurons. The study will focus on EGR1 and Synaptotagmin IV, both of which have been linked to different stages of learning and memory. ? ? ?
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