The goal: To prepare oligonucleotide probes that are coupled to fluorochromes to enter the cell and detect intracellular mRNAs in living cells with single copy sensitivity. This application is dedicated to increase in signal to noise ratio by factors exceeding 20 fold over existing technologies, focused mainly on background reduction approaches. 1. To synthesize fluorochrome coupled oligonucleotide probes using a phosphorothioate linkage to enter the cell and hybridize to RNA. We will then optimize the detection of the hybridized probes by initially using the site of highest signal in the nucleus: where the RNA is being transcribed. Various cellular models will be used with a dynamic range of four orders of magnitude; the highest can generate as many as 48,000 fluorochromes at the transcription site. We will then proceed to quantitatively test improvements in sensitivity by using models with progressively fewer targets. 2. To design a fluorochrome-quencher combination that will result in fluorescence only when the probe hybridizes to its target. We will design a library of chemical compounds to screen using a high-throughput probe fluorescence assay. 3. To design imaging hardware and software to filter out the background generated by unhybridized probes leaving only the hybridized fluorescent signals. 4. To multiplex the probes with different fluorochromes so that we will be able to detect multiple RNA species in the same cell. Ultimately, the approach can be formatted for use in living tissue. ? ? ?
| Noordermeer, Daan; de Wit, Elzo; Klous, Petra et al. (2011) Variegated gene expression caused by cell-specific long-range DNA interactions. Nat Cell Biol 13:944-51 |
| Wang, Donghai; Pezo, Rossanna C; Corner, Georgia et al. (2010) Altered dynamics of intestinal cell maturation in Apc1638N/+ mice. Cancer Res 70:5348-57 |
