Rho ( �� ) Analysis to Dissect RNA Folding and Assembly Pathways

Area of Science:

• Molecular Biology
• Biophysics
• Structural Biology

Background:

• RNA folding is complex, influenced by modular structures and energetics.
• Traditional methods like phi (ϕ) analysis rely on internal comparisons, limiting insights.
• Understanding RNA conformational pathways is crucial for function and regulation.

Purpose of the Study:

• Introduce Rho (ρ) analysis, a novel method to systematically dissect RNA conformational pathways.
• Utilize isolated RNA contacts as external standards for deeper mechanistic insights.
• Apply ρ analysis to understand RNA folding pathways and RNA-guided DNA recognition.

Main Methods:

• Developed Rho (ρ) analysis using isolated RNA secondary or tertiary contacts as external standards.
• Compared kinetic effects of mutations on folding rates with thermodynamic stability of isolated contacts.
• Applied ρ analysis to Tetrahymena group I intron folding and CRISPR-Cas12a RNA-guided DNA recognition.

Main Results:

• ρ analysis provides insights into when RNA interactions form relative to the rate-limiting transition state.
• Demonstrated ρ analysis on Tetrahymena group I intron, revealing multiple folding pathways and flux modulation.
• Observed substantial phi (ϕ) values in CRISPR-Cas12a binding, indicating a late transition state, and quantified mismatch penalties.

Conclusions:

• Rho (ρ) analysis is a general, straightforward framework for probing RNA conformational pathways and function.
• The method is applicable both in vitro and within cellular environments.
• Findings advance understanding of RNA folding, dynamics, and specificity in biological systems.