Single Molecule Studies of TYMV RNA Secondary Structure: the AFM Approach.

In biological systems, RNA can assume a variety of different roles: the genome of viruses, the messenger of the genetic information in the cells, the structural and functional constituent of molecular machineries (in the ribosome). A single-stranded RNA molecule (ssRNA) can assume a huge variety of different conformations through the combination of different secondary structure elements, like duplex tracts, unstructured tracts, hairpins, bulges, internal loops and junctions. These elements can interact with each other leading to complex tertiary structures, so it is possible to say that ssRNA is more similar to proteins than to DNA (1). There exist several different stable conformations for a RNA molecule of defined sequence, but probably only one of many structures of similar stability is selected by nature for its function. It is thus necessary to improve the currently available techniques for the study of the secondary and tertiary structure of RNA. Even though computational predictive tools are available, the need of studying the response of RNA to its cellular environment motivates the development of experimental analysis methods. Electron Microscopy (EM) can bring an important contribution to this subject as it allows characterizing directly the shape and conformation of each individual molecule. Atomic Force Microscopy (AFM), more than EM, has been widely utilized to characterise nucleic acids (2) and to study their interactions with other specimens, like proteins (3), as it allows to work in quasi-native conditions. Even though the AFM cannot visualize all the single structural elements of the RNA molecules adsorbed on a surface, due to it is resolution limitations, it can be expected that it can visualize the molecules with sufficient detail to characterize the shape of the structural domains (probably comprising more than one hairpin). It is thus possible to reach a mesoscopic structural characterization of the molecules, and to correlate it with the environmental conditions. We studied the conformations of the 3’-end of the RNA genome of Turnip Yellow Mosaic Virus (TYMV), already characterized by means of computational, physical and chemical studies (4). Our AFM micrographs revealed structures characterized by separated domains, segregated by tracts of unstructured ssRNA. Through image processing operations, we could map the position of the different domains along the molecules and their folding state in correlation to the different environmental conditions. The data showed that a progressive increment of temperature and, counter intuitively, of the concentration of Mg(II) causes a progressive unfolding of the structures. In conclusion, we have developed a new procedure for mapping the secondary structure of single molecules of RNA from their AFM images. Our operational method (AFM imaging and image analysis) could also be extended to other similar tasks that require the structural characterization of surface adsorbed molecules. (1) Batey, R. T., R. P. Rambo, et al. (1999). Angew. Chem. Int. Ed. Engl. 38(16): 2326-2343. (2) Zuccheri, G., A. Scipioni, et al. (2001). Proc. Natl. Acad. Sci. USA 98(6): 3074-3079. (3) Rivetti, C., S. Codeluppi, et al. (2003). J. Mol. Biol. 326(5): 1413-26. (4) Hellendoorn, K., A. W. Mat, et al. (1996). Virology 224(1): 43-54.