Single molecule studies of RNA secondary structure: AFM of TYMV viral RNA

Abstract

Nowadays, the development of experimental procedures for the determination of the secondary structure of RNA molecules is taking advantage of the novel single-molecule probing and imaging techniques. We report a method for the mapping of the secondary structure of RNA molecules spread on a flat surface by means of the atomic force microscope. Globular domains comprising groups of RNA secondary and tertiary structure elements separated by unstructured domains can be discerned in the micrographs and their position along the molecule contour can be measured directly on unstained specimens. We have analyzed the morphology of a population of single molecules of 3' fragments of the Turnip Yellow Mosaic Virus RNA shorter than 1 kb in different temperature and electrolytic conditions. We found a satisfying agreement of the shape of the imaged structures with previously available evidence. The method we have developed can be used to map also different types of RNA molecules and has the advantage of showing the distribution of the single molecule conformations within the population.

Conserved Pseudoknots in lncRNA MEG3 Are Essential for Stimulation of the p53 Pathway

“…Having established that MEG3 compacts in a specific manner in vitro and that it forms a functionally important pseudoknot in vitro and in vivo , we attempted to visualize this lncRNA by single-particle 3D imaging. For our study, we used atomic force microscopy (AFM), a technique used previously to image other multi-domain structured RNAs and to monitor RNA conformational changes (García-Sacristán et al., 2015, Giro et al., 2004, Hansma et al., 1996, Lyubchenko et al., 2011, Schön, 2016, Yu et al., 2015).…”

A magnesium-induced RNA conformational switch at the internal ribosome entry site of hepatitis C virus genome visualized by atomic force microscopy

“…The nanometer resolution of this technique is optimal for the visualization of nucleic acid molecules ( 41 – 45 ), including RNA and RNA-protein complexes ( 46 – 49 ). AFM is increasingly being used in virology ( 50 – 53 ) and low-resolution images of the whole HCV genomic RNA have been published ( 54 , 55 ). However, AFM has not yet been applied to the structural characterization of either viral 5′ UTR regions or viral/cellular IRES elements.…”

Evaluation of chemical labeling strategies for monitoring HCV RNA using vibrational microscopy

“…Recently, it was extended to the investigation of RNA secondary structure and viral RNAs. [60][61][62][63][64][65][66][67][68] We employed AFM to investigate the bulk structural effects of modification to aaU-HCV RNA and CN-HCV RNA relative to HCV RNA. These samples are the most amenable to study with AFM because the nature of their modifications imply that they would experience the largest structural perturbations and were, therefore, likely to be within the resolution limits of AFM.…”