By Isabella Saira, Fall 2020.

Understanding the structure and function of the fundamental molecules required for life is a task scientists have been trying to achieve for generations. Part of the daunting task of decoding who we are at the molecular level is finding ways to visualize microscopic building blocks, including RNA. RNA, known as ribo-nucleic acid, is present in all cells; its principal role is to serve as a messenger and carry information from DNA related to protein synthesis. RNA is a large scale molecule in comparison to other essential molecules, like carbohydrates and lipids, therefore finding an effective way to image it has proven difficult. 

RNA imaging has progressed in incredible ways throughout the last fifty years. The first attempts to image RNA molecules occurred in the early 1970’s, when scientists focused on small scale RNA base-pairing attractions [1]. Although these processes were not consistently all-encompassing or incredibly accurate, their use in RNA analysis was integral to the future development of NMR based analysis. Only a few years later, scientists applied X-ray diffraction in order to visualize RNA, a technique previously used to image DNA [1]. The first major publication on X-ray diffraction in the context of RNA and RNA spectroscopy was published in 1977 [2]. However, there were still many technological limitations, and it was difficult to image RNA on a two-dimensional scale as opposed to a one-dimensional scale due to the lack of advanced technology. In the 1980’s, scientists achieved two-dimensional NMR studies which were soon followed by detailed structural studies [3]. After this point, NMR became a novel and effective tool that allowed scientists to begin to more easily image RNA, which in turn has led to significant insights into the molecule’s structure and function. 

The process of NMR spectroscopy is the same regardless of the scientific context it is applied in. The technique was widely used in the field of physics prior to its application in  nucleic acid visualization. Essentially, the process involves directing radio waves at a target molecule and subsequently measuring the changes in the magnetic field around the atoms, which is known as the nucleic magnetic resonance [4]. NMR spectroscopy is primarily used for structural analysis and determination of molecules, including the analysis of nonstandard geometries like bent helices, non-Watson Crick base pairings, and coaxial stacking. In addition, the technique can be used to determine local structures within the molecule like glycosidic bond angles, dihedral angles, sugar pucker confirmations, imino proton resonances (or lack thereof), and so on [5]. Despite the accuracy and usefulness of NMR spectroscopy, the technique still has some limitations with respect to the size of the molecule that one can image. Due to its relatively large size, until recently, scientists were able to image only parts of the RNA molecule at once. 

New advances in imaging large scale RNA molecules have revolutionized the way we think about the structure and function of RNA and of large molecules in general. University of Maryland scientists recently developed a method for imaging three-dimensional RNA molecules [6]. Previously, using NMR spectroscopy, scientists were able to analyze small RNA molecules (>35 nucleotides) by hitting them with radio waves. When these molecules are hit, fluorine essentially “lights up” by giving off a signal which can be subsequently measured. However, as molecules get bigger, the fluorine does not give off a signal strong enough for an accurate measurement of the spectra [6]. Previous studies had established that fluorine produces strong signals when next to carbon-13 isotopes. Thus, these scientists developed a method to switch the C-12 atom already in RNA to a C-13 atom and put a F-19 atom right next to the newly switched C-13. To do this, they used RNA from HIV, which had been previously imaged, to test their method. Once they were successful, they moved on to Hepatitis-B RNA, a molecule double the size of HIV RNA. Through the use of their newly developed method, the scientists were able to identify places on HepB RNA where small molecules may bind. Knowing these locations is incredibly useful for disease treatment, as it helps scientists develop more specific and targeted drugs. The specificity granted by this method is especially important concerning drug development for another reason; since RNA changes shape frequently, it is particularly difficult to visualize, and developing drugs that don’t affect typical and beneficial cell function is challenging [5]. For example, if scientists design a drug that targets RNA in one structural state without realizing the target RNA actually changes shape, the drug may subsequently become ineffective. 

Using NMR to image large RNA molecules will remove the uncertainty of shape change and allow for more specific and accurate drugs to treat disease. Thus, the discovery made by these researchers is integral to drug design both in vitro and in vivo and will revolutionize the way we analyze large scale RNA molecules. NMR spectroscopy for nucleic acid imaging has come a long way and, as it continues to be refined, we will be able to further understand the intricacies of these molecules and how they can be used for more specialized and accurate treatments. 

1. Carlomagno, Teresa. “Present and Future of NMR for RNA–Protein Complexes: A Perspective of Integrated Structural Biology.” Journal of Magnetic Resonance. Academic Press, March 20, 2014. https://www.sciencedirect.com/science/article/pii/S1090780713002620. 
2. Thomas, G. J., and A. H.-J. Wang. “Laser Raman Spectroscopy of Nucleic Acids.” SpringerLink. Springer, Berlin, Heidelberg, January 1, 1988. https://link.springer.com/chapter/10.1007/978-3-642-83384-7_1. 
3. Barnwal, Ravi P, Fan Yang, and Gabriele Varani. “Applications of NMR to Structure Determination of RNAs Large and Small.” Archives of biochemistry and biophysics. U.S. National Library of Medicine, August 15, 2017. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5555312/. 
4. Jeener, Jean. “Jeener, Jean: Reminiscences about the Early Days of 2D NMR.” Wiley Online Library. American Cancer Society, March 15, 2007. https://onlinelibrary.wiley.com/doi/abs/10.1002/9780470034590.emrhp0087. 
5. Fürtig, Boris, Christian Richter, Jens Wöhnert, and Harald Schwalbe. “NMR Spectroscopy of RNA.” Chemistry Europe. John Wiley & Sons, Ltd, September 26, 2003. https://chemistry-europe.onlinelibrary.wiley.com/doi/full/10.1002/cbic.200300700. 
6. Becette, Owen B., Guanghui Zong, Bin Chen, Kehinde M. Taiwo, David A. Case, and T. Kwaku Dayie. “Solution NMR Readily Reveals Distinct Structural Folds and Interactions in Doubly 13C- and 19F-Labeled RNAs.” Science Advances. American Association for the Advancement of Science, October 1, 2020. https://advances.sciencemag.org/content/6/41/eabc6572.