Biological structures whose sizes lie between those of individual macromolecules and cellular organelles (a few nm to 100 nm) are not readily studied with existing technologies. Thus, while vast inventories of RNA and protein sequences and structures are being catalogued, a quantitative cellular context for these structures is lagging. Understanding this context would have great value, because it would allow us to know how collections of individual macromolecules assemble into the dynamic machines that form a living cell. Once these interactions are revealed, a new picture of the cell and its disease states is sure to emerge. The long-term goal of this project is to develop new biomolecule tagging methodologies that will enable the construction of 3D maps of RNA and proteins in cells using a combination of intracellular fluorescence imaging, electron tomography (ET) and electron energy loss spectroscopy (EELS). The tags will consist of RNA or peptide sequences that are engineered to catalyze the formation of inorganic nanoparticles (""""""""materials ribozymes and enzymes""""""""). Once genetically encoded in cells as RNA concatemers and protein chimeras, these sequences will be able to catalyze the formation of inorganic nanoparticles (4 nm - 10 nm diameter) inside living cells exclusively at the site of interest. The long-term goal will be implemented by identifying a library of unique clonable materials ribozymes and enzymes. The nanoparticles they generate will have one or more of 5 desired properties: (1) Nanoparticles that can be synthesized in live cells;(2) Nanoparticles that can be synthesized in cells that have been prepared by vitrification and freeze-substitution in cold acetone;(3) Luminescent nanoparticles;(4) Shape or size distinct nanoparticles;and (5) Nanoparticles with distinct compositions that can be resolved using EELS. Collectively these tags will enable a highly multiplexed approach to imaging cellular biomolecules, conceivably allowing the visualization of tens to hundreds of biomolecules in a single tomogram. A series of well-defined aims demonstrate how the proposed library of nanoparticle tags may be created and validated.
The aims are to: (1) isolate, through biomolecule in vitro selection methods, materials enzymes and ribozymes and screen them in vitro against a specific set of chemical criteria to determine their likelihood of functioning in vivo;(2) Use the sequences that satisfy the criteria from aim 1 to create RNA concatemers and peptide chimeras in a model bacterial system, and test them in vitro and in vivo for their ability to be used as nanoparticle tags;and finally (3) Demonstrate the multiplexing capabilities of genetically encoded nanoparticle tags by localizing two proteins simultaneously with ET. Public Health Relevance: The long-range goal of this project is to generate a complete 3D map of the spatial arrangement of RNA and proteins in a cell. This goal will be accomplished through the implementation of a new concept in cellular imaging, in which inorganic nanoparticles are used to tag biomolecules in vivo for visualization with 3D electron tomography. Mapping these multi-component biomolecule interactions will provide an unprecedented glimpse of cellular biochemistry and the discrete molecular changes that differentiate normal processes from disease processes.
| Chen, Songye; McDowall, Alasdair; Dobro, Megan J et al. (2010) Electron cryotomography of bacterial cells. J Vis Exp : |
| Carter, Carly Jo; Ackerson, Christopher J; Feldheim, Daniel L (2010) Unusual reactivity of a silver mineralizing peptide. ACS Nano 4:3883-8 |
| Pilhofer, Martin; Ladinsky, Mark S; McDowall, Alasdair W et al. (2010) Bacterial TEM: new insights from cryo-microscopy. Methods Cell Biol 96:21-45 |
