RNA Splicing at the Single Molecule Level
- Electronic Properties of Molecules Used for Opto-electronic Devices
- Emission Properties of Single Molecules and Aggregates
- RNA Splicing at the Single Molecule Level
- Optical Properties of Dye-Labeled DNA Gold Nanoparticle Constructs
- Development of Novel Probes for Intra-Cellular Studies
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Instrumentation:
- Single Molecule Fluorescence Microscopy
Pre-mRNA splicing is an essential and precisely regulated process in gene expression that is performed by a large and dynamic ribonucleoprotein complex known as the spliceosome. It involves the removal of non-coding regions of the pre-mRNA (introns) and the joining of the remaining exons to form a functional mRNA product from which protein translation occurs. In collaboration with the groups of Bruce Armitage (Chemistry) and Javier Lopez (Biology) we are studying this phenomenon on the single molecule level using fluorescence microscopy. This requires the fluorescent labeling of the RNA exon regions of the yeast pre-mRNA using novel protein nucleic acid (PNA)-based probes to follow the in vitro splicing kinetics using single molecule FRET. We use RT-PCR to show that the high-FRET states monitored dynamically are indeed due to the correctly spliced mRNA product, demonstrating that the probes are non-intrusive. The ability to follow the splicing of individual pre-mRNA molecules is valuable for elucidating the dynamic changes in composition and structure of the spliceosome that accompany catalysis and exploring the basis of splicing fidelity. 
