| In vivo techniques to investigate nucleic acid metabolism, distribution and function have thus far been the exclusive domain of protein biology. In order to fully grasp the diverse roles of nucleic acids in living cells, it is essential to be able to track and image the distribution of DNA and RNA molecules in their native context. Currently, only green fluorescent protein and its variants permit this. A nucleic acid equivalent of fluorescent proteins remains an elusive goal. Nucleic acid probes such as molecular beacons, fluoresce only upon hybridization to their nucleic acid complements. They offer great promise in visualizing nucleic acid dynamics in living cells. My broad aim is to directly image RNA and DNA molecules in living cells, while following their origins, distributions, localizations and fates, temporally and spatially. Experiments in living cells with molecular beacons lead to a startling observation that a priori made them unsuitable for the detection of cytoplasmic RNA, since they are completely sequestered in the nucleus. This sequestration is independent of the fluorophore, sequence or phosphodiester backbone utilized. Thus, to image the distribution of RNA throughout the cell, nuclear sequestration of nucleic acid probes needs to be overcome. My general solution to this problem is to exploit the existing machinery that eukaryotic cells have evolved to export different macromolecules from the nucleus to the cytoplasm. Three approaches were pursued, utilizing either the protein export, mRNA export or the tRNA export pathways. This study lead to the discovery of new aspects of nucleocytoplasmic transport. Prior to this work, it was believed that only correctly processed and trimmed tRNA could be exported from the nucleus. However, cytoplasmic localization has been maintained by fusing in vitro constructed tRNA via a maleimide bond to molecular beacons. tRNA-molecular beacons are exported from the nucleus intact, and are able to bind specifically to their targets. Furthermore the tRNA pathway is able to export numerous variants of RNA fused to tRNA, counter to currently understood mechanisms of tRNA export. Thus by developing new methods for in vivo visualization of RNA, new aspects of nucleocytoplasmic transport have been unmasked. |
