Single molecule approaches for studying spliceosome assembly and catalysis
Biochemistry & Molecular Pharmacology
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. GSBS Student Publications. https://doi.org/10.1007/978-1-62703-980-2_17. Retrieved from https://escholarship.umassmed.edu/gsbs_sp/2006