Selective 2'-hydroxyl acylation analyzed by protection from exoribonuclease

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) is a key method for RNA structure and dynamics.
• Traditional SHAPE has limitations in resolving RNA structure at the 5' and 3' ends due to primer extension detection.
• Primer extension detection in SHAPE is more complex than the chemical modification step.

Purpose of the Study:

• To develop a novel method for directly quantifying RNA adducts.
• To overcome the limitations of primer extension in SHAPE analysis.
• To analyze RNA structure and dynamics with improved resolution, particularly at termini.

Main Methods:

• Quantification of covalent adducts in RNA by adduct-inhibited exoribonuclease degradation.
• Utilizing RNase R, a 3'-->5' exoribonuclease, which is blocked by RNA 2'-O-adducts.
• Analysis of the thiamine pyrophosphate (TPP) riboswitch aptamer domain structure.

Main Results:

• Developed RNase-detected SHAPE (selective 2'-hydroxyl acylation analyzed by protection from exoribonuclease) for direct adduct quantification.
• Identified significant changes in local nucleotide dynamics and global RNA structure of the TPP riboswitch upon ligand binding.
• Discovered a single nucleotide bulge register shift distant from the binding site that stabilizes the ligand-bound structure.

Conclusions:

• RNase-detected SHAPE offers a more direct and potentially simpler method for RNA structure analysis compared to primer extension.
• This method overcomes resolution issues at RNA termini, providing more complete structural information.
• RNase-detected SHAPE is broadly applicable for structural analysis of small noncoding RNAs and RNAs with critical terminal structures.