Precise Measurement of the Relative Position of RNA Dimers within Virus-Like Particles using 2-Color 3D Super-Resolution Fluorescence Microscopy

Abstract

A long-standing question in retrovirus biology is how RNA genomes are spatially organized within virions. Here, we explore the relative 3D spatial distribution of RNA dimers (1) within ∼130-nm diameter virus-like particles (VLPs) with single-RNA sensitivity (2) using the double-helix microscope. The double-helix microscope, a modification of the standard epifluorescence microscope, is capable of precise and accurate three-dimensional (3D) localization of fixed fluorescent single molecules throughout a 2 μm-thick focal volume (3). Furthermore, the use of a sophisticated registration algorithm, based upon a locally weighted quadratic transformation function, enables accurate co-localization of yellow and red fluorescent emitters with <10 nm registration error in 3D (4). We routinely localize fluorescently tagged RNA coat protein-clusters (via Bgl- and MS2-based fluorescent protein labeling) to a 3D precision of 10-15 nm within the VLPs. By systematically varying the location of these coat protein-clusters along the length of the RNAs, we elucidate the spatial organization of packed RNA dimers inside these VLPs.1. Nikolaitchik, O. A., et al. 2013. PLoS Pathog. 9, e1003249.2. Chen, J., et al. 2009. Proc. Natl. Acad. Sci. USA. 106, 13535-13540.3. Backlund, M. P., et al. 2012. Proc. Natl. Acad. Sci. USA. 109, 19087-19092.4. Gahlmann, A., et al. 2013. Nano Lett. 13, 987-993.