Mimicking Ribosomal Vectorial Unfolding of RNA Pseudoknot in a Protein Channel

Pseudoknots are a fundamental RNA tertiary structure found in almost all classes of RNAs. They not only play a key role in diverse biological mechanisms, but also provide an excellent model for understanding RNA folding/unfolding processes involved in their functions. Molecular force spectroscopic approaches such as optical tweezers can track the pseudoknot's unfolding intermediate states by pulling the molecule from both 5’ and 3’-ends, but the pseudoknot unfolding kinetic pathway may be different from that in vivo, which proceeds in the translation direction from the 5’ to 3’ end. Here we developed a ribosome-mimicking, nanopore pulling assay for dissecting the vectorial unfolding mechanism of pseudoknots. The unfolding pathway of pseudoknot in the nanopore, either from the 5’ to 3’ end or in the reverse direction, can be controlled by a DNA lead that is attached to the pseudoknot at the 5’ or 3’ ends. The different nanopore conductance between DNA and RNA translocation serves as a marker for the position and structure of the unfolding RNA in the pore. With this design, we identify that the pseudoknot unfolding is a two-step, multi-state, metal ion-regulated process, and along different pathways dependent on the pulling direction. Notably, the unfolding in both directions are rate-limited by the unzipping of the first helix domain, which is Helix-1 in the 5’→3’ direction and Helix-2 in the 3’→5’ direction, suggesting that the initial unfolding step in either pulling direction needs to overcome an energy barrier contributed by the non-canonical triplex base-pairs and coaxial stacking interactions for the tertiary structure stabilization. These findings provide new insights into RNA vectorial unfolding mechanism. Potentially, the nanopore assay could be adapted to study different pseudoknots and riboswitches, shedding light on their biological functions including frameshifting and anti-virus drug design.