Aaron Hoskins, PhD
New Approaches for Imaging RNAs by Fluorescence Microscopy
University of Wisconsin - Madison
Assembly of ribonucleoproteins (RNPs) from their constituent RNAs and proteins is a complicated process but critical for a number of cellular activities including splicing, translation, and telomere formation. We are interested in using fluorescence microscopy to study RNP assembly pathways in real time both in vitro and in live cells. However, to achieve this goal new methods for preparing and imaging fluorescent RNAs and RNPs need to be developed. Here we present three approaches that can be used to prepare fluorescent RNAs and RNPs for biochemical analysis in the test tube, in whole cell lysates, or in living cells. In the test tube, we have developed a method using deoxyribozymes (DNAzymes) to site-specifically install fluorophores into large RNA transcripts. The resulting RNAs can be used for a number of applications including single molecule microscopy. In whole cell lysates, we can now attach fluorophores to various RNPs and purify them in a matter of seconds in situ on a single molecule microscope. This approach will give biochemists access to low abundance RNPs and a means to biochemically interrogate them. Finally, we have developed novel RNA aptamers that can bind commercially-available benzylguanine fluorophores. These aptamers should facilitate delivery of high quantum yield and photoswitchable organic fluorophores to specific RNAs in live cells to enable super resolution fluorescence microscopy of RNP assembly dynamics. In sum, our new tools for targeting RNAs and RNPs with fluorophores represent enabling technologies for biochemical elucidation of RNA biology.