Figure 1. Designed cotranscriptional RNA structures

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Figure 1. Designed cotranscriptional RNA structures Figure 1. Designed cotranscriptional RNA structures. (A) Secondary structures of the ‘direct’ RNA switch by Isambert ... Figure 1. Designed cotranscriptional RNA structures. (A) Secondary structures of the ‘direct’ RNA switch by Isambert and coworkers (10). The branched structure was formed almost exclusively by transcription from a DNA template. After renaturation by heating/cooling, the RNA equilibrated to approximately equal amounts of the branched and the rod-like structures. Therefore, the two structures are similar in thermodynamic stability, but the branched structure is kinetically stable when generated cotranscriptionally. The bases that form the central stem in the rod-like structure are shown in red for reference. (B) Hairpin/G4 structures by Sugimoto and coworkers (11). Hairpin formation is favored cotranscriptionally, but it slowly and spontaneously relaxes to a G4 structure. The bases involved in G4 are indicated in red. Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Nucleic Acids Res, gkz364, https://doi.org/10.1093/nar/gkz364 The content of this slide may be subject to copyright: please see the slide notes for details.

Figure 2. Proposed secondary structures of the metastable RNAs Figure 2. Proposed secondary structures of the metastable RNAs. (A) The majority of the L+U transcript adopts the ... Figure 2. Proposed secondary structures of the metastable RNAs. (A) The majority of the L+U transcript adopts the branched structure because stem I is formed first cotranscriptionally. The branched structure is metastable (kinetically stable) and is robust enough to maintain its structure for 17 h at 4°C. Renaturation by heating and cooling transforms most of the RNA to the rod-like structure with stems III and IV. The Broccoli aptamer (green) can bind DFHBI-1T and fluoresce only in the rod-like structure. (B) Variants of L+U intended to increase the relative stability of the rod-like structure compared to the branched structure. Introduced mutations are indicated by red circles. L+U-stemIV+1 harbors a single mutation near the 3′ end that extends stem IV by 1 bp. wMT8 contains a C-A mismatch within stem I to destabilize the branched structure, with a compensatory mutation near the 3′ end to stabilize stem IV, therefore, the rod-like structure. A similar set of mutations were introduced in L+U-mm6 which contains a mismatch at a different location within stem I. Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Nucleic Acids Res, gkz364, https://doi.org/10.1093/nar/gkz364 The content of this slide may be subject to copyright: please see the slide notes for details.

Figure 3. Characterization of metastable and thermodynamically stable RNA structures. (A) Fluorescence of the ... Figure 3. Characterization of metastable and thermodynamically stable RNA structures. (A) Fluorescence of the metastable RNAs (500 nM) generated by in vitro transcription with and without renaturation after heating. The solutions contain 5 μM DFHBI-1T. The blank contains the buffer and DFHBI-1T. Fluorescence values were normalized to that of the untreated wMT8 with DFHBI-1T (1.0). The data are averages of three replicate samples with the error bars representing S.D. (B) Fluorescence of the same samples in (A) after 17 h kept at 4°C. (C) Native gel electrophoresis of in vitro transcribed wMT8. An aliquot was kept on ice (left lane), and another aliquot was renatured by heating to 75°C followed by slow cooling. Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Nucleic Acids Res, gkz364, https://doi.org/10.1093/nar/gkz364 The content of this slide may be subject to copyright: please see the slide notes for details.

Figure 4. Catalytic relaxation of wMT8 by a trigger RNA Figure 4. Catalytic relaxation of wMT8 by a trigger RNA. (A) Mechanism of catalytic transformation of wMT8 from the ... Figure 4. Catalytic relaxation of wMT8 by a trigger RNA. (A) Mechanism of catalytic transformation of wMT8 from the metastable to the thermodynamically stable structure by a trigger RNA (c4+10 shown). The sequence complementary to the trigger RNA is highlighted in yellow, with the toehold region marked with a red line. The internal toehold region that is transiently activated by the trigger RNA hybridization is highlighted in blue. (B–D) RNA triggered catalytic wMT8 relaxation monitored via Broccoli-DFHBI-1T fluorescence. All reactions contained 1.0 μM wMT8, 10 μM DFHBI-1T. Trigger RNA concentration was 100 nM unless specified otherwise. (B) Variation of toehold length in cN<sub>toe</sub>+10 where N<sub>toe</sub> = 0–9. The numbers indicate N<sub>toe</sub>. NC: no catalyst. (C) Varying the invading strand length (N<sub>inv</sub> = 8, 9, 10) for N<sub>toe</sub> = 3, 4, or 5. (D) wMT8 response to different concentrations of c4+8. Data shown are averages of two replicates. Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Nucleic Acids Res, gkz364, https://doi.org/10.1093/nar/gkz364 The content of this slide may be subject to copyright: please see the slide notes for details.

Figure 5. Metastable RNA sensors designed to detect microRNA sequences Figure 5. Metastable RNA sensors designed to detect microRNA sequences. The part of miRNA sequence (pink) that ... Figure 5. Metastable RNA sensors designed to detect microRNA sequences. The part of miRNA sequence (pink) that hybridize with the sensor is underlined. The sensor sequence complementary to the targeted microRNA is highlighted in yellow. The graphs indicate the fluorescence responses of the sensors (1 μM). Trigger RNA concentration was 100 nM. NC: no catalyst. (A) miR-21. (B) miR-122. Data shown are averages of three replicates. Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Nucleic Acids Res, gkz364, https://doi.org/10.1093/nar/gkz364 The content of this slide may be subject to copyright: please see the slide notes for details.