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. 2016 Feb;22(2):225–236. doi: 10.1261/rna.052233.115

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© 2016 Rodgers et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society

This article is distributed exclusively by the RNA Society for the first 12 months after the full-issue publication date (see http://rnajournal.cshlp.org/site/misc/terms.xhtml). After 12 months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.

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FIGURE 1. — Model RNA design and smFRET data. (A) Model of the yeast U2 snRNA. The U2 core RNA used in smFRET assays (nt 34–120, displayed as stem IIa/b) is boxed. Light gray boxes represent the branch point recognition sequence and Sm protein binding site. (B) Design of a smFRET reporter of stem II conformation. Fluorophores were placed to produce a high FRET signal during stem IIc formation when they come into close proximity. (C, top panel) Example smFRET data from the WT U2 core RNA showing anti-correlated changes in Cy3 donor (green) and Cy5 acceptor (red) fluorescence indicative of FRET. (Bottom panel) Calculated E_FRET from the data in the top panel shows fluctuations between mid and high E_FRET values. (D) Distribution of E_FRET values for the WT U2 core RNA (N = 100 molecules). (E) Distribution of E_FRET values after extension by hybridization to a complementary DNA oligo (N = 81). (F) Distribution of E_FRET values for a stem IIa RNA (N = 105). (G) Distribution of E_FRET values for a stem IIc RNA (N = 72). The data in E and G could be fit by a single Gaussian distribution (black lines) centered at 0.06 and 0.99, respectively.