Direct visualization of the native structure of viroid RNAs at single-molecule resolution by atomic force microscopy

Viroids are small infectious, non-protein-coding circular RNAs that replicate independently and, in some cases, incite diseases in plants. They are classified into two families: Pospiviroidae, composed of species that have a central conserved region (CCR) and replicate in the cell nucleus, and Avsunviroidae, containing species that lack a CCR and whose multimeric replicative intermediates of either polarity generated in plastids self-cleave through hammerhead ribozymes. The compact, rod-like or branched, secondary structures of viroid RNAs have been predicted by RNA folding algorithms and further examined using different in vitro and in vivo experimental techniques. However, direct data about their native tertiary structure remain scarce. Here we have applied atomic force microscopy (AFM) to image at single-molecule resolution different variant RNAs of three representative viroids: potato spindle tuber viroid (PSTVd, family Pospiviroidae), peach latent mosaic viroid and eggplant latent viroid (PLMVd and ELVd, family Avsunviroidae). Our results provide a direct visualization of their native, three-dimensional conformations at 0 and 4 mM Mg²⁺ and highlight the role that some elements of tertiary structure play in their stabilization. The AFM images show that addition of 4 mM Mg²⁺ to the folding buffer results in a size contraction in PSTVd and ELVd, as well as in PLMVd when the kissing-loop interaction that stabilizes its 3D structure is preserved.