Detecting RNA-RNA interactions using psoralen derivatives.

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• Psoralens are tricyclic compounds known to intercalate into DNA and RNA.
• Upon UV irradiation, psoralens form covalent cross-links between pyrimidine bases on adjacent nucleic acid strands.
• These cross-links can occur within double-stranded regions or at double-stranded-single-stranded boundaries.

Purpose of the Study:

• To describe a protocol for utilizing psoralens to detect RNA-RNA base-pairing interactions.
• To highlight the utility of 4 aminomethyl trioxsalen (AMT) as a water-soluble psoralen derivative for this purpose.
• To detail the method for detecting and reversing psoralen-induced RNA cross-links.

Main Methods:

• Utilizing 4 aminomethyl trioxsalen (AMT) to induce cross-linking in RNA within cells or extracts.
• Irradiating samples with long-wavelength (365-nm) UV light to facilitate psoralen cross-linking.
• Analyzing cross-linked RNA by observing reduced mobility in polyacrylamide gel electrophoresis.
• Reversing cross-links by exposure to short-wavelength (254 nm) UV light.

Main Results:

• Psoralen treatment and UV irradiation effectively create covalent cross-links in RNA.
• Cross-linked RNA molecules exhibit decreased migration in polyacrylamide gels compared to uncross-linked controls.
• The cross-linking process is reversible, allowing for the recovery of intact RNA.

Conclusions:

• 4 aminomethyl trioxsalen (AMT) is an effective reagent for detecting RNA-RNA interactions.
• The described method provides a reliable way to identify base-paired regions within RNA molecules.
• Psoralen-mediated cross-linking offers a valuable tool for studying RNA structure and interactions in various biological contexts.