AFM study of TYMV viral RNA secondary structure.

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. Even though there exist several methodology to investigate ssRNA secondary structure, the need of studying the response of RNA to its cellular environment motivates the development of new experimental analysis methods. Atomic Force Microscopy (AFM) can bring an important contribution to this subject as it allows characterizing directly the shape and conformation of each individual molecule in quasi-native 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 studies1. Our AFM micrographs revealed molecules characterized by separated structured 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. In conclusion, we have developed a new procedure for mapping the secondary structure of single molecules of RNA from their AFM images. 1Hellendoorn, K., A. W. Mat, et al. (1996) Secondary structure model of the coat protein gene of turnip yellow mosaic virus RNA: long, C-rich, single-stranded regions, Virology 224(1): 43-54.