Fig. 2: Distinguishing between LMW RNA tertiary structures with MspA.
a The scheme of nanopore trapping/translocation using MspA. Current fluctuations between trapping and translocation reveal the RNA identity. A tRNA was employed as an example. b Representative RNA molecules studied in this manuscript. Five types of RNAs, including miRNA (single stranded, 22 nt), overhanged siRNA (double-stranded, 21 bp), blunt siRNA (double-stranded, 21 bp), tRNA (L shaped, 76 nt), and 5 S rRNA (Y shaped, 120 nt) were investigated. The measurements were carried out as described in Methods. MiRNA (has-miR-21), overhanged siRNA (SiFoxA1), blunt siRNA (luciferase siRNA), or tRNA (tRNAphe) were added to cis with a final concentration of 200 nM for each analyte. E.coli 5 S rRNA was added to cis with a final concentration of 10 nM. c Representative traces of successive translocations of miRNA (orange), overhanged siRNA (blue), blunt siRNA (green), tRNA (red), or 5 S rRNA (purple). The open pore current (\({I}_{o}\)) is marked with dashed lines. d Zoom-in views of representative translocation events from marked triangles of corresponding traces. Translocations of different RNA types result in highly distinguishable events features. MiRNA gives rise to fast spiky events. Overhanged siRNA produces two-step events. Blunt siRNA and tRNA both generate two types of events, termed type 1 and type 2. 5 S rRNA gives rise to three types of signals, which the most characteristic type is shown in the figure. The \(\% {I}_{b}\) refers to the first-level blockade amplitude which is defined as marked in d. e A scatter plot of \(\% {I}_{b}\) versus \({t}_{{off}}\) for five RNA samples. Events from five types of RNAs are clearly distinguishable. f The corresponding event histogram of \(\% {I}_{b}\) of different RNA types. Black lines are Gaussian fittings to the data. g A representative trace during simultaneous sensing of siRNA, tRNA, and 5 S rRNA. Different RNA types (overhanged siRNA: 25 nM; blunt siRNA: 10 nM; tRNA: 400 nM; 5 S rRNA: 30 nM) were simultaneously added to cis side. Characteristic events from different RNA types are clearly recognized from the trace, which are marked with blue, green red, or purple bars respectively.
