(Supplied by the Applicant): This application addresses broad Challenge Area (06) Enabling Technologies and specific Challenge Topic 06-GM-102* .Chemist/biologist collaborations facilitating tool development. The objective of this grant is to develop fluorescent chemical tags that both have high photon counts and are cell permeable and soluble to enable single-molecule detection in cells. Single-molecule imaging can now be carried out in vitro and has provided a breakthrough for understanding the molecular mechanisms of complex biological assemblies because it allows the dynamics of individual complexes to be directly observed--but in vivo single-molecule imaging has only been carried out by a handful of leading experts in the field. In our opinion a major barrier to routine single-molecule imaging in cells is the difficulty of labeling proteins selectively in cells with suitable fluorophores. The fluorescent proteins do not have high enough photon counts to be readily detected with single molecule resolution in cells, and the organic fluorophores with high photon counts developed for in vitro biophysics do not behave well in the cell.
Here, we propose that chemical tags including our commercial TMP-tag can provide the combination of genetic encoding and an organic fluorophore label needed for single-molecule imaging in cells. We have assembled an interdisciplinary team of a chemical biologist expert in chemical tags (PI Cornish), a physical chemist skilled in fluorescence microscopy (co-PI Kaufman), a leading biochemist studying the mechanism of the spliceosome reaction (co-PI Moore), and a cell biologist who is a pioneer in the application of state-of-the-art microscopy to cell motility (co-PI Sheetz) to meet this objective. We propose to meet this objective by (1) challenging the chemical tags for single-molecule imaging of the spliceosome in yeast cell extracts, (2) developing fluorescent chemical tags suitable for single-molecule imaging in cells, and (3) developing chemical tags with specialized properties for high-resolution imaging.
Showing the most recent 10 out of 11 publications