Lipid Vesicle Characterization and Encapsulation for smFRET Experiments
Black, William A.
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In eukaryotes, a protein-RNA complex called the spliceosome carries out gene splicing, a type of RNA processing. Although much is known about the spliceosome, it still remains unclear which component catalyzes the splicing reaction, RNA or protein. Similarities between RNA components of the spliceosome and self-splicing catalytically active RNAs have led to the hypothesis that the RNA element of the spliceosome catalyzes splicing. Previous experiments have indicated that the RNA component of the spliceosome forms three distinct conformations; however, it’s unknown which specific conformation corresponds to each stage of splicing. smFRET experiments of active spliceosomes could be used to investigate the conformational changes of the spliceosome during splicing. In this work, yeast splicing extract was isolated and splicing activity was confirmed through a splicing reaction using a radio-labeled oligonucleotide. Lipid vesicles were extruded in order to immobilize the spliceosome for smFRET experiments. Vesicles were characterized with dynamic light scattering and microscopy. In order to investigate vesicle encapsulation, an oligonucleotide labeled with two fluorpohores was utilized. smFRET experiments confirmed the encapsulation of the oligonucleotide, based on the presence of photobleaching of both fluorophores and a high FRET state. These experiments have made significant progress towards studying active yeast spliceosomes on the single molecule level.