Uncovering the Structural Landscape of Heat-Induced Riboswitch RNA Unfolding Using Native Variable-Temperature ESI...

Area of Science:

• Biochemistry
• Structural Biology
• Molecular Biophysics

Background:

• RNA structures denature with increasing temperature, leading to loss of secondary structure.
• Conformations of RNA at temperatures above base-pairing disruption are poorly understood.

Purpose of the Study:

• To map the stepwise unfolding and refolding of a riboswitch aptamer using multiple techniques.
• To understand RNA conformational dynamics at elevated temperatures.

Main Methods:

• Variable-temperature nanoelectrospray ionization ion mobility mass spectrometry (VT-nanoESI-IM-MS)
• Imino-proton nuclear magnetic resonance (NMR) spectroscopy
• UV absorbance spectroscopy

Main Results:

• Three distinct thermal regimes of RNA unfolding were identified.
• Region I (25-55 °C): Local melting and reduced phosphate accessibility.
• Region II (60-75 °C): Global loss of secondary structure and increased charge.
• Region III (80-90 °C): Formation of compact, non-native conformers with shielded phosphates.

Conclusions:

• RNA melting can result in compact, non-native conformations, not just complete disorder.
• These alternative conformations limit charge uptake due to shielded phosphate groups.
• Findings expand understanding of RNA conformational dynamics under thermal stress.