Effects of flanking regions on HDV cotranscriptional folding kinetics

Hepatitis delta virus (HDV) ribozyme performs the self-cleavage activity through folding to a double pseudoknot structure. The folding of functional RNA structures is often coupled with the transcription process. In this work, we developed a new approach for predicting the cotranscriptional folding kinetics of RNA secondary structures with pseudoknots. We theoretically studied the cotranscriptional folding behavior of the 99-nucleotide (nt) HDV sequence, two upstream flanking sequences, and one downstream flanking sequence. During transcription, the 99-nt HDV can effectively avoid the trap intermediates and quickly fold to the cleavage-active state. It is different from its refolding kinetics, which folds into an intermediate trap state. For all the sequences, the ribozyme regions (from 1 to 73) all fold to the same structure during transcription. However, the existence of the 30-nt upstream flanking sequence can inhibit the ribozyme region folding into the active native state through forming an alternative helix Alt1 with the segments 70–90. The longer upstream flanking sequence of 54 nt itself forms a stable hairpin structure, which sequesters the formation of the Alt1 helix and leads to rapid formation of the cleavage-active structure. Although the 55-nt downstream flanking sequence could invade the already folded active structure during transcription by forming a more stable helix with the ribozyme region, the slow transition rate could keep the structure in the cleavage-active structure to perform the activity.