Measuring Charge Transport Properties in Single and Double-Stranded RNA Oligonucleotides at the Nanoscale

RNA oligonucleotides and their biophysics are an essential part of therapeutics, sensing, and forensics applications. They have gained importance in the research focus in recent years for several reasons. Not only do several pathogens (e.g., SARS-CoV2) have RNA genomes, but many therapeutic¹, biotechnological², and modern molecular technologies (e.g., Gene editing³, gene silencing⁴) use RNA oligonucleotides at their core.<br/>Single-molecule electrical techniques have allowed studying the charge transfer process in biomolecules with unprecedented resolution in the last decade⁵. In the biomolecular electronics discipline, the electronic and charge-transport (CT) properties of short oligonucleotides (dsDNA &DNA: RNA hybrid) have been reported extensively⁶. But despite its biophysical and biological importance, the single-molecule electronic properties of double-stranded(ds)RNA and single-stranded (ss) RNA have not been studied to that extent.<br/>Biomolecular electronics, the discipline studying the charge transfer process in biomolecules, has the potential to describe the interaction between electronic materials and biological systems (RNAs) at molecular interfaces. We use the Scanning tunneling microscope-assisted break junction method (STM-BJ)⁷to study charge transport in short oligonucleotides by reproducible conductance histograms related to their biomolecular structure and conformations. Each biomolecule will be confined within a nanometric gap at a well-defined molecular orientation to measure its electrical properties. Herein, we measured, for the first time, the conductance of individual dsRNA molecules and compared it with the conductance of DNA: RNA hybrids⁸. 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. In our next study, we have demonstrated base-mediated charge transport in specific single-stranded 5 and 10 base pairs(bp) RNA oligonucleotide sequences. We found base stacking is the main factor for getting a probable conductance value for those spontaneously formed RNA oligonucleotide junctions. By comparing with their DNA counterpart, we have found that single-stranded DNA for both 5 and 10 bp sequences are less conductive than RNA due to a lack of base stacking in them.<br/>These results pave the way for measuring various biomolecular interactions at a single-molecule level. In the future, we will measure the conductance of a protein (Argonaute)– RNA complex. This can give us a better understanding of the formation of the RISC(RNA-induced silencing complex) complex and allow the study of the thermodynamics and kinetics of gene expression at the individual complex level. Lastly, we can use these fundamental results for designing next-generation smart biomaterials and biosensors that may address improved biological performances and advanced health monitoring.<br/><br/>References:<br/>1. C. F. Bennett and E. E. Swayze, Annu. Rev. Pharmacol. Toxicol., 2010, 50, 259–293.<br/>2. J. A. Doudna and E. Charpentier, Science, 2014, 346, 6213.<br/>3. D. B. T. Cox, R. J. Platt, and F. Zhang, Nat. Med., 2015, 21, 121–131.<br/>4. Zhang, Y., W. Dubitzky, et al., RNA-induced Silencing Complex (RISC), in Encyclopedia of Systems Biology, Editors. 2013, Springer New York: New York, NY. p.1876-1876.<br/>5. K. G. G. P. Arachchillage, S. Chandra, A. Piso, T. Qattan and J. M. A. Vivancos, J. Mater. Chem. B, 2021, 9(35), 6994–7006<br/>6. Li, Yuanhui, et al. "Comparing charge transport in oligonucleotides: RNA: DNA hybrids and DNA duplexes." The journal of physical chemistry letters 7.10 (2016): 1888-1894.<br/>7. B. Xu and N. J. Tao, Science, 2003, 301, 1221–1223.<br/>8. Chandra, Subrata, et al. "Single-molecule conductance of double-stranded RNA oligonucleotides." Nanoscale 14.7 (2022): 2572-2577.