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A Novel Saturation Mutagenesis Approach: Single Step Characterization of Regulatory Protein Binding Sites in RNA Using Phosphorothioates
JoVE Journal
Genetics
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JoVE Journal Genetics
A Novel Saturation Mutagenesis Approach: Single Step Characterization of Regulatory Protein Binding Sites in RNA Using Phosphorothioates
DOI:

11:49 min

August 21, 2018

Chapters

  • 00:04Title
  • 00:30Generation of a Library of Mutants – Doping DNA Template with Non-wild Type Nucleotides
  • 01:20Synthesis of RNA
  • 05:24Protein Binding Reaction and Separation of Bound RNA
  • 08:05Analysis of Iodine-cleaved Phosphorothioate Products for Detection of Mutant Nucleotide Positions
  • 09:07Results: Characterization of Protein Binding Site in RNA
  • 10:22Conclusion

Summary

Automatic Translation

Proteins that bind specific RNA sequences play critical roles in gene expression. Detailed characterization of these binding sites is crucial for our understanding of gene regulation. Here, a single-step approach for saturation mutagenesis of protein-binding sites in RNA is described. This approach is relevant for all protein-binding sites in RNA.

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