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Screening helix-threading peptides for RNA binding using a thiazole orange displacement assay
Malathy Krishnamurthy1, Nicole T Schirle, Peter A Beal
1Department of Chemistry, University of California, Davis, One Shields Avenue, Davis, CA 95616, USA.
Bioorganic & Medicinal Chemistry
|September 16, 2008
Summary
A new assay using fluorescent intercalator displacement (FID) measures RNA-binding helix-threading peptides (HTPs) without radioactivity. This method reveals RNA binding depends on peptide structure, not macrocycle size.
Area of Science:
- Biochemistry
- Molecular Biology
- Structural Biology
Background:
- Helix-threading peptides (HTPs) are studied for RNA binding.
- Traditional methods for studying RNA-peptide interactions often require radiolabeling and gel electrophoresis.
Purpose of the Study:
- To adapt the fluorescent intercalator displacement (FID) assay for studying RNA-binding HTPs.
- To determine structure-activity relationships for HTPs binding to RNA.
- To assess the sensitivity and accuracy of the FID assay compared to existing methods.
Main Methods:
- Adaptation of the thiazole orange-based fluorescent intercalator displacement (FID) assay.
- Application of the FID assay to a library of helix-threading peptides.
- Comparison of FID assay results with quantitative ribonuclease footprinting data.
Main Results:
- The FID assay is highly sensitive for detecting HTP-binding RNAs.
- Binding affinity data from the FID assay closely matched quantitative ribonuclease footprinting results.
- RNA binding by HTPs was found to be dependent on peptide sequence, alpha-amino acid stereochemistry, and cyclization.
- Macrocyclic ring size did not affect RNA binding for the analyzed penta-, tetra-, and tri-peptides.
Conclusions:
- The FID assay provides a sensitive, non-radioactive method for studying RNA-peptide interactions.
- Peptide sequence, stereochemistry, and cyclization are critical determinants of RNA binding affinity for HTPs.
- The FID assay is a valuable tool for defining structure-activity relationships in peptide nucleic acid interactions.
