Fig. 2.

Nanopore snapshot for T2 RNA pseudoknot formation. a Stepwise multi-level nanopore current signature generated by a complex probe under the +120 mV/t _fold = 10 s/−60 mV protocol (the probe was trapped at  + 120 mV, the folding time t _fold was 10 s, and the folding structure was unfolded at −60 mV). b Model cartoons showing the entire trapping–folding–unfolding procedure revealed by the signature in a: translocation of the 5ʹ DNA tag (A, Level-1), disruption while translocating T2 RNA (B, Level-2), translocation and immobilization of the 3ʹ DNA tag (C, Level-1), folding of the pseudoknot from the single-stranded RNA on the trans side (D) for a pre-defined folding time (t _fold = 10 s), unfolding of the folding structure under back-pulling at −60 mV while the 3ʹ DNA tag remains in the pore (E, Level-3), translocation of the partially unfolded T2 RNA through the pore (F, Level-4), and a fully disrupted probe pulled out of the pore and back to the cis solution (G). The time from the beginning of negative voltage (−60 mV) application to the block end is the unfolding duration of an RNA folding state (τ), during which the folding structure was disrupted