Probing RNA Conformations Using a Polymer–Electrolyte Solid-State Nanopore

Nanopore systems have emerged as a leading platform for the analysis of biomolecular complexes with single-molecule resolution. The conformation of biomolecules, such as RNA, is highly dependent on the electrolyte composition, but solid-state nanopore systems often require high salt concentration to operate, precluding analysis of macromolecular conformations under physiologically relevant conditions. Here, we report the implementation of a polymer–electrolyte solid-state nanopore system based on alkali metal halide salts dissolved in 50% w/v poly(ethylene) glycol (PEG) to augment the performance of our system. We show that polymer–electrolyte bath governs the translocation dynamics of the analyte which correlates with the physical properties of the salt used in the bath. This allowed us to identify CsBr as the optimal salt to complement PEG to generate the largest signal enhancement. Harnessing the effects of the polymer–electrolyte, we probed the conformations of the Chikungunya virus (CHIKV) RNA genome fragments under physiologically relevant conditions. Our system was able to fingerprint CHIKV RNA fragments ranging from ∼300 to ∼2000 nt length and subsequently distinguish conformations between the co-transcriptionally folded and the natively refolded ∼2000 nt CHIKV RNA. We envision that the polymer–electrolyte solid-state nanopore system will further enable structural and conformational analyses of individual biomolecules under physiologically relevant conditions.


Table of Contents
Supporting Figure 9. Population scatter plots for events recorded when dsDNA was translocated into either a lithium halide or sodium halide electrolyte bath containing PEG 35K. All nanopipettes were filled with 0.3 nM of the 4.8 kbp dsDNA diluted in 0.1M KCl. The nanopipettes were dipped into baths containing 0.1 M lithium halide +50% (w/v) PEG 35K or 0.1 M sodium halide +50% (w/v) PEG 35K electrolyte bath. The metal halide used is indicated on the upper right corner of each graph. Translocation of the dsDNA was performed by applying a voltage of -500 mV to cause the dsDNA to migrate from the nanopipette to the bath. The graphs are organised such that the halide atomic number/mass increase from left to right (from fluoride to iodide). N is the number of data plotted. Figure 10. Population scatter plots for events recorded when dsDNA was translocated into either a potassium halide or caesium halide electrolyte bath containing PEG 35K. All nanopipettes were filled with 0.3 nM of the 4.8 kbp dsDNA diluted in 0.1M KCl. The nanopipettes were dipped into baths containing 0.1 M potassium halide +50% (w/v) PEG 35K or 0.1 M caesium halide +50% (w/v) PEG 35K electrolyte bath. The metal halide used is indicated on the upper right corner of each graph. Translocation of the dsDNA was performed by applying a voltage of -500 mV to cause the dsDNA to migrate from the nanopipette to the bath. The graphs are organised such that the halide atomic number/mass increase from left to right (from fluoride to iodide). N is the number of data plotted.

Supporting
Supporting Figure 11. The linear regression plots of the current and the dwell time against the lattice energy of the alkali metal halides. The average dwell time (A) and current peak maxima (B) for translocation events for dsDNA translocated into 0.1M alkali metal halide +50% (w/v) PEG 35K electrolyte baths plotted against the lattice energy for the salts. Linear regressions were performed on both data set, the R 2 indicates the coefficient of determination.
Supporting Figure 12. Nanopore analysis of the 500 bp dsDNA translocated into a PEG electrolyte bath containing different halide salts. (A) 1 µg of the gel extracted 500 bp dsDNA was analysed by gel electrophoresis with a 0.8% SYBR safe incorporated agarose gel. A DNA ladder (GeneRuler 100 bp (SM0241, Thermo Fisher)) was run alongside. The extracted sample migrated at ~ 500 bp relative to the DNA ladder. The 500 bp dsDNA fragment was isolated and purified from the same DNA ladder. Supporting Figure 13. Analysis of in vitro transcribed CHIKV RNA fragments by 2% denaturing formaldehyde MOPS agarose gel electrophoresis. CHIKV RNA fragments were resolved by electrophoresis on a 2% denaturing formaldehyde MOPS agarose gel, the RNA ladder used is the RNA markers (G3191, Promega). Two bands were observed in the 1987 nt denatured RNA samples, this could be due to the incomplete denaturation of the RNA and the formation of secondary RNA structures.
Supporting Figure 14. The translocation of 1987 nt RNA fragments into 0.1 M KCl bath, in the absence of PEG 35K. A voltage of -500 mV was used to cause the RNA to migrate from the nanopipette to the 0.1 M KCl bath. Despite some peaks were observed, no translocation peaks were called with our custom written nanopore events analysis MATLAB script, due to the stringent threshold detection level.
Supporting Figure 15. The translocation trace of the co-transcriptionally folded and the natively refolded 1987 CHIKV RNA. -500 mV was used to cause the RNA to translocate from nanopipette to the PEG 35K electrolyte bath.

Supporting Tables
Supporting Table 1 contains the experimental lattice energy value in kJ/mol we used in Figure 3 and Supporting Figure 11. The values can be found in the CRC handbook of chemistry and physics 1 .

Electrolyte bath preparation
The

RNA synthesis and quality control
The RNA used in this study was generated by in vitro transcription. The CHIKV infectious clone and sub-genomic replicons used in this study were derived from the LR2006_OPY1 La Reunion island isolate of the ECSA genotype (accession number DQ443544) 3

RNA synthesis related sequence
Below shows the sequence of the three DNA templates used to generate the RNA fragments: CHIKV 318 nt

Scanning electron microscopy
The nanopores of the nanopipettes were imaged by scanning electron microscopy (Leo 1530 FEG-SEM; Zeiss). Nanopipettes were first sputter coated with a gold layer of a few nanometres in thicknesses. The nanopipettes were then mounted onto the sample holder and tilted to an angle of nearly 90 for imaging. The nanopipettes were imaged at between 2 and 3 kV at a working distance of 5 mm and below at an aperture size of 30.00 m using an InLens detector.