Measuring intramolecular connectivity in long RNA molecules using two-dimensional DNA patch-probe arrays.

Area of Science:

• Molecular Biology
• Biophysics
• Genomics

Background:

• RNA molecules adopt complex three-dimensional structures crucial for their function.
• Understanding RNA intramolecular connections is vital for deciphering RNA structure-function relationships.
• Existing methods often rely on computational folding models, which may not capture the full complexity of RNA conformations in solution.

Purpose of the Study:

• To develop and validate a novel DNA-array-based method for inferring RNA intramolecular connections and their prevalences.
• To investigate the coexistence of multiple RNA structures in solution.
• To examine the impact of RNA modifications and strand types on RNA connectivity.

Main Methods:

• A DNA-array-based patch-probe technique was employed to perturb and map RNA connections.
• DNA oligonucleotide "patches" were used to disrupt specific RNA interactions.
• Microarrays with DNA oligonucleotide "probes" recorded perturbation patterns to infer connectivity.

Main Results:

• The patch-probe method successfully inferred intramolecular connections in satellite tobacco mosaic virus (STMV) RNA without prior folding models.
• Results revealed long-range connections consistent with known structures and identified competing connections, suggesting multiple coexisting RNA structures.
• The study demonstrated that pseudouridine substitution and strand type (plus vs. minus) significantly alter RNA connectivity.
• A simplified version validated a predicted connection in bacteriophage MS2 RNA.

Conclusions:

• The DNA patch-probe method provides a powerful, model-free approach to map RNA connectivity and prevalence in solution.
• Multiple RNA structures with distinct connectivity patterns can coexist, offering insights into RNA dynamics.
• RNA modifications and strand polarity play critical roles in shaping RNA structural ensembles.