Fig. 1.

Single-molecule detection of RNA folding in a protein nanopore. a 2D (left) and 3D (right) structures of the pseudoknot from the 5ʹ end gene 32 messenger RNA of bacteriophage T2 (PDB id: 2tpk), which contains two helices, H1 (red) and H2 (blue), and two loops (green), L1 and L2. The base triple interactions between loops and helices (identified by RNAview⁷⁰) and the coaxial stacking between the two helices are illustrated. b Nanopore detection of T2 RNA folding states and the folding pathway. The detection was facilitated by a complex probe containing a chimera of the 5ʹ-DNA tag, T2 RNA, and 3ʹ-DNA tag. The 3ʹ end is attached to streptavidin (SA) to immobilize the probe. Upon immobilization, the T2 RNA starts to fold from the single-stranded form in the trans solution. After a pre-defined folding time, the folding process is terminated at an intermediate state (B) or the pseudoknot structure (C) by using a negative voltage that disrupts the folding structure (D). The unfolding signatures reveal the folding state (B and C) and unfolding state (A). The folding time-dependent population of the identified folding states allows establishment of the pseudoknot folding pathway