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Single-molecule conductance of double-stranded RNA oligonucleotides.

RNA oligonucleotides are crucial for a range of biological functions and in many biotechnological applications. Herein, we measured, for the first time, the conductance of individual double-stranded (ds)RNA molecules and compared it with the conductance of single DNA : RNA hybrids. The average conductance values are similar for both biomolecules, but the distribution of conductance values shows an order of magnitude higher variability for dsRNA, indicating higher molecular flexibility of dsRNA. Microsecond Molecular Dynamics simulations explain this difference and provide structural insights into the higher stability of DNA : RNA duplex with atomic level of detail. The rotations of 2'-OH groups of the ribose rings and the bases in RNA strands destabilize the duplex structure by weakening base stacking interactions, affecting charge transport, and making single-molecule conductance of dsRNA more variable (dynamic disorder). The results demonstrate that a powerful combination of state-of-the-art biomolecular electronics techniques and computational approaches can provide valuable insights into biomolecules' biophysics with unprecedented spatial resolution.

Other parts of the STM cell were cleaned by Acetone (HPLC plus), and Ethyl Alcohol (pure), were purchased from Sigma Aldrich. Before each experiment, Au single crystal was electropolished in 0.1 M sulfuric acid (Sigma Aldrich) using two thin gold wire loops as electrodes. Au single crystal was placed on top of one gold wire loop (working electrode), and it was connected to the DC power supply. The other gold wire loop (counter electrode) was placed closer to the single crystal (preferably less than 0.5 cm) and connected to the same DC supply. We used the DC power supply to achieve about 10V and 1 Amp current. The current was applied for about 30 seconds until we observed the Au single crystal had oxidized to form an orange layer on top. Then, the oxidized Au single crystal was rinsed in Milli-Q water multiple times. After that, the rinsed Au single crystal was placed into 1M HCl solution and rinsed again in Milli-Q water until we observed the pure gold color back, as previously described. 1 Finally, the cleaned Au single crystal was flame annealed. 2 Na2HPO4 and NaH2PO4 were purchased from Sigma-Aldrich to prepare 100 mM pH 7.4 phosphate buffer with Milli-Q water. Then the buffer solution was filtered with 20 nm pore size filters (Whatman Anotop 25 plus). The filtered buffer solution was then ready to be used in experiments. Au STM tips were cutted from Au wire (0.25 mm diameter) for the conductance measurements, and then covered with Apiezon wax to isolate them from any leakage currents.

STM-Break junction measurements
All experiments were performed at room temperature. Experiments were done using a Molecular Imaging Pico-STM head connected to a Digital Instruments Nanoscope IIIa controller. In the STM-BJ method, the STM tip was repeatedly brought into contact with the substrate and then pulled away (Tapping method). 3 Experiments were performed with a 100 mv bias voltage applied between the tip and the sample. This process forms a tip-substrate gap that is narrow enough to be bridged by a molecule. In this method, a large number of individual molecules and their conformations can be probed by repeating the experiment many times. Thousands of currentdistance traces were recorded during each experiment. Some of these traces show steps, which indicate the presence of oligonucleotide junctions between tip and substrate.
Before measuring oligonucleotide sequences, a control experiment in a phosphate buffer solution was performed. Then, small volumes of RNA: DNA hybrid or dsRNA were injected into the cell to achieve ~ nM final concentrations for the conductance measurements.
Tris (2-carboxyethyl) phosphine (TCEP) was used to reduce the disulfide bond. TCEP selectively and completely reduces even the most stable water-soluble alkyl disulfides over a wide pH range. This treatment produces free thiol groups from the oligonucleotide sequence, which will eventually bind the gold electrodes.

Labview Software
A customized LabView (National Instruments) program was used to control the movement of the STM tip during the measurements 4 . This program performs a logarithmic binning of each recorded current-distance trace and then selects those whose residual values meet the specified criteria. All the filtered curves were added together to obtain a semi-logarithmic conductance histogram, which revealed the most probable conductance of the specific molecular junction.

Data Treatment
We have accumulated thousands of current-distance traces to obtain conductance histograms. After saving 5000-10000 traces, selectivity (defined as the number of curves containing significant "steps") ranged from less than 1% in blank and control experiments to 5 to 10% in experiments with nucleic acids. For dsRNA, the number of accumulated traces was 338 (9.12% of the total captured, fig. S5). For DNA: RNA, 257 traces were taken to build the histogram (5.11% of the total captured, Fig 1. c). Then we have applied Gaussian fitting upon the histogram peaks to get the most probable conductance value. To get the FWHM value in fig 1.d we did the normalization of the conductance histograms after subtracting the baseline from the actual histogram. For a Gaussian-shaped conductance histogram, full width at half maximum (FWHM) is the width of a line shape at half of its maximum amplitude.

Modeling of RNA and DNA sequences
SimRNA software 5 was utilized to model RNA sequences in dsRNA. A DNA strand in RNA:DNA hybrid was constructed by replacing 2'-OH group by 2'-H group in ribose rings using VMD package 6 . The topology and coordinate files for dsRNA and RNA:DNA hybrid were prepared using tleap module in AMBER 20 7 . The system was neutralized using Na + and Clions and excess ions (0.1 M) were added to mimic the experimental conditions. The force fields bsc0χOL3 8 used for RNA and bsc0 9 for DNA is a part of ff14SB 10 . Using TIP3P water molecules, the dsRNA and DNA:RNA systems were solvated up to 8Å from any of the solute atoms 11 . The number of water molecules in both systems were ∼3,700.

MD simulation protocol
The water molecules and ions in the solvated systems were minimized in 10,000 steps of the steepest descent, and in 50,000 steps of the conjugate gradient with 50 kcal/mol restraint on the solute atoms. The system was heated from 0K to 300K in 50 ps. Next, a 100 ps of restrained (0.5 kcal/mol on solute atoms) MD simulations was performed to equilibrate each system. The production MD simulations were performed for 1 μs using CUDA version of pmemd 12 utilizing a GPU acceleratation 13,14 of AMBER 20 7 . The Particle Mesh Ewald (PME) method was utilized for calculating the contributions from the nonbonded interactions with a cutoff of 10Å. SHAKE algorithm was used to treat the bonds involving light (hydrogen) atoms. Next, the unrestrained MD simulations were performed in an NPT-ensemble with the time step of 2 fs. A constant external pressure of 1 atm was maintained using Bendersen weak-coupling barostat with the time constant of 0.5 ps 15 . The MD simulation temperature (300K) was maintained using a Bendersen thermostat with a time constant of 4 ps. The MD trajectories were saved every 20 ps.

Analysis of MD simulation output
The root mean square deviation (RMSD) was calculated for all atoms of the solute using the RMSD Trajectory Tool in VMD 2 . RMSDs were calculated for the backbone heavy atoms of RNA/DNA (P, O5′, C5′, C4′, C3′, and O3′). All the frames (structure snapshots) were superimposed with the structure corresponding to 500 ns (initial structure) after removing the water molecules from the MD output. The snapshots depicting the averaged structures (shown in the background in the Supplementary Movie1) were calculated using the Tcl script in Tk Console in the VMD. The endto-end distance X was calculated as a distance between the first and last (11 th ) P-atoms in the passenger strand in dsRNA or DNA strand in DNA:RNA hybrid. The total number of base pairs and base stackings were calculated using Barnaba software 12 . Bases were classified as stacked if (� � and � � > 2Å) and ( or < 2.5Å) and (� � < 40°). Here, = � 2 + 2 , whereand -axis are in the plane of the base and -axis is normal to the -plane, is distance between the centers of mass of the two bases, and is the angle between the normal vectors of the two bases 12 . All the non-stacked bases are considered base-paired if � � < 60° and there exists at least one hydrogen (H) bond. We assume that the H-bond D-H…A between the hydrogen donor atom (D) and acceptor atom (A) is formed if the donor-acceptor distance is less than the 3.3Å cutoff and the bond angle is larger than the 140°cutoff 12 . We also analyzed the torsion angles in RNA and DNA backbones, in particular, torsion angle δ formed by C5'-C4'-C3'-O3' atoms, and glycosidic angle χ formed by O4'-C1'-N9-C4 and O4'-C1'-N1-C2 atoms for purine and pyrimidine bases.

Calculation of thermodynamic state functions
Van der Waals interaction energies between two strands in the duplex (dsRNA or RNA:DNA hybrid) were computed utilizing MMPBSA pairwise energy decomposition analysis using a reported procedure 17,18 implemented in MMPBSA.py program 19 in AMBERTools 7 . For this calculation, structure snapshots were extracted from the last 500 ns part of MD trajectories taken at a time interval of 5 ns.  The movie shows conformational fluctuations in dsRNA (left) and DNA:RNA hybrid (right) as observed in MD simulations. Transient structures are superposed with the average structure displayed in the background. The MD simulation runs were carried out in explicit water with 0.1 M NaCl, but the movie shows only nucleic acids for clarity. Each duplex displays the secondary structure and chemical bonds with "NewCartoon" and Bonds representations implemented in VMD (the blue color is for dsRNA and red color is for DNA:RNA). Shown are 500-ns long MD simulation runs. The length of the movie is 24 s. The movie is played 48,000,000 times slower than the computational experiment.