Single molecule approaches for studying spliceosome assembly and catalysis
Department of Biochemistry and Molecular Pharmacology
Single molecule assays of splicing and spliceosome assembly can provide unique insights into pre-mRNA processing that complement other technologies. Key to these experiments is the fabrication of fluorescent molecules (pre-mRNAs and spliceosome components) and passivated glass slides for each experiment. Here we describe how to produce fluorescent RNAs by splinted RNA ligation and fluorescent spliceosome subunits by SNAP-tagging proteins in cell lysate. We then depict how to passivate glass slides with polyethylene glycol for use on an inverted microscope with objective-based total internal reflection fluorescence (TIRF) optics. Finally, we describe how to tether the pre-mRNA onto the passivated slide surface and introduce the SNAP-tagged cell lysate for analysis of spliceosome assembly by single molecule fluorescence.
DOI of Published Version
Methods Mol Biol. 2014;1126:217-41. doi: 10.1007/978-1-62703-980-2_17. Link to article on publisher's site
Methods in molecular biology (Clifton, N.J.)
Anderson EG, Hoskins AA. (2014). Single molecule approaches for studying spliceosome assembly and catalysis. Morningside Graduate School of Biomedical Sciences Student Publications. https://doi.org/10.1007/978-1-62703-980-2_17. Retrieved from https://escholarship.umassmed.edu/gsbs_sp/2006