Transcription reshapes RNA hairpin folding pathways revealed by all-atom molecular dynamics simulations

The divergence in folding pathways between RNA co-transcriptional folding (CTF) and free folding (FF) is crucial for understanding dynamic functional regulation of RNAs. Here, we developed a simplified all-atom molecular dynamics framework to systematically compare the folding kinetics of an RNA hairpin (PDB:1ZIH) under CTF and FF conditions. By analyzing over 800 microseconds of simulated trajectory, we found that despite convergence to identical native conformations across CTF simulations (with varied transcription rates) and FF simulations, they exhibit distinct preferences for the folding pathways defined by the order of base-pair formation. Conformational space analysis shows that CTF biases the folding pathway by adopting more compact conformations than FF. Our findings provide atomic-scale insights into how temporal-spatial coupling of transcription and folding diversifies RNA folding dynamics.