Figure 1. Concept of poly(A) tail labeling for translation and localization analyses of reporter mRNAs. Azido-modified ... Figure 1. Concept of poly(A)

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Figure 1. Concept of poly(A) tail labeling for translation and localization analyses of reporter mRNAs. Azido-modified ... Figure 1. Concept of poly(A) tail labeling for translation and localization analyses of reporter mRNAs. Azido-modified adenosine nucleotides are enzymatically attached at the poly(A) tail of in vitro transcribed reporter mRNAs (luciferase or eGFP) using yeast poly(A) polymerase. (A) Translation analysis. (B) Intracellular labeling. 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 Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Nucleic Acids Res, Volume 47, Issue 7, 06 February 2019, Page e42, https://doi.org/10.1093/nar/gkz084 The content of this slide may be subject to copyright: please see the slide notes for details.

Figure 2. Enzymatic addition of modified adenosines to the 3′-end of short test RNA. (A) Scheme illustrating 3′-end ... Figure 2. Enzymatic addition of modified adenosines to the 3′-end of short test RNA. (A) Scheme illustrating 3′-end modification of test RNA with 2′-N<sub>3</sub>-2′-dATP or 3′-N<sub>3</sub>-2′,3′-ddATP using yeast PAP followed by click reaction with DBCO-biotin. Using 2′-N<sub>3</sub>-2′-dATP, multiple 2′-N<sub>3</sub>-2′-deoxyadenosine nucleotides were added at the poly(A) tail and then reacted quantitatively to the corresponding click product. Using 3′-N<sub>3</sub>-2′,3′-ddATP, a single 3′-N<sub>3</sub>′-2′,3′-dideoxyadenosine nucleotide is added and reacted during click reaction. (B, C) MS analysis of adenylated and clicked RNA after digestion and dephosphorylation to ribonucleosides: 2′-click-2′-dA 2 (calculated mass of [C<sub>49</sub>H<sub>64</sub>N<sub>13</sub>O<sub>11</sub>S]<sup>+</sup> = 1042.4563, found: 1042.4565) or 3′-click-2′,3′-ddA 4 (calculated mass of [C<sub>49</sub>H<sub>64</sub>N<sub>13</sub>O<sub>10</sub>S]<sup>+</sup> = 1026.4614, found: 1026.4616), respectively. (D) HPLC analysis of adenylated test RNA before and after click reaction. Samples were prepared as in B, C. (E) PAGE analysis of intact modified test RNAs (15% PAA gel, 12 W, 3 h, rt). SYBR™ Gold staining (Invitrogen) is shown. 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 Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Nucleic Acids Res, Volume 47, Issue 7, 06 February 2019, Page e42, https://doi.org/10.1093/nar/gkz084 The content of this slide may be subject to copyright: please see the slide notes for details.

Figure 3. In vitro fluorescence labeling of poly(A) tail-modified reporter mRNAs. (A) Concept for poly(A) tail ... Figure 3. In vitro fluorescence labeling of poly(A) tail-modified reporter mRNAs. (A) Concept for poly(A) tail modification of mRNAs with 2′-N<sub>3</sub>-2′-dATP using yeast PAP followed by click reaction using DBCO-SRB. (B, C) Luciferase (GLuc, CLuc) and eGFP mRNAs were analyzed by PAGE (7.5% PAA gel, 10 W, 1 h, rt) and visualized using SRB fluorescence (λ<sub>ex</sub> = 532 nm) and SYBR™ Gold (Invitrogen) staining. 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 Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Nucleic Acids Res, Volume 47, Issue 7, 06 February 2019, Page e42, https://doi.org/10.1093/nar/gkz084 The content of this slide may be subject to copyright: please see the slide notes for details.

Figure 4. Stability and translational efficiency of poly(A) tail-modified luciferase mRNAs. (A) Scheme illustrating ... Figure 4. Stability and translational efficiency of poly(A) tail-modified luciferase mRNAs. (A) Scheme illustrating translation assay in mammalian cells of poly(A)-modified luciferase mRNAs followed by luminescence analysis of secreted luciferases. (B) Western blot analysis of GLuc protein in the supernatant of cells transfected with indicated GLuc-mRNA constructs. With the cell lysate of corresponding samples, GAPDH protein level from the respective cell lysate is shown as control. (C) Translational efficiency of differently modified GLuc-mRNAs relative to co-transfected CLuc-mRNA. The GLuc luminescence was referenced to the luminescence from ARCA-capped CLuc-mRNA and then normalized to ARCA-GLuc mRNA after 24 h (≙ 100%). Data and error bars show average and standard deviation of three independent experiments measured in duplicate. Statistical comparisons performed by two-tailed t-test with P < 0.01 (**) and P < 0.001 (***), statistical comparisons to ApppG and untransfected cells are not shown). (D) Comparison of GLuc- and CLuc-mRNA levels by qPCR at 24 h after transfection for different combinations of modified poly(A) tails, as indicated in the legend. The GLuc or CLuc mRNA level was quantified by qPCR and compared to the ß-actin-mRNA level by generating ΔCt = Ct<sub>(target)</sub>-Ct<sub>(reference)</sub>. Data and error bars show averages and standard deviations of two independent experiments with two technical replicates measured in duplicate. Abbreviations: Ct: cycle threshold, target: GLuc- or CLuc-mRNA, reference: ß-actin-mRNA, ApppG / ARCA: capped 5′-end, N<sub>3</sub>: azido-modified poly(A) tail, click: clicked poly(A) tail). 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 Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Nucleic Acids Res, Volume 47, Issue 7, 06 February 2019, Page e42, https://doi.org/10.1093/nar/gkz084 The content of this slide may be subject to copyright: please see the slide notes for details.

Figure 5. Poly(A) tail-modified eGFP-mRNA is a translationally active mRNA. (A) Scheme illustrating transfection of ... Figure 5. Poly(A) tail-modified eGFP-mRNA is a translationally active mRNA. (A) Scheme illustrating transfection of HeLa cells with poly(A) tail-modified mRNAs (ARCA-N<sub>3</sub>, ARCA-click) or with unmodified control (ARCA, ApppG) and following translation into protein. (B) The eGFP and GAPDH protein level of different cell samples was analyzed on western blots. (C) Confocal microscopy shows eGFP fluorescence after transfection with indicated modified mRNAs. 24 h after transfection, cells were fixed for imaging. Scale bars are 20 μm. 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 Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Nucleic Acids Res, Volume 47, Issue 7, 06 February 2019, Page e42, https://doi.org/10.1093/nar/gkz084 The content of this slide may be subject to copyright: please see the slide notes for details.

Figure 6. eGFP-mRNA with azido-modified poly(A) tail can be labeled in living cells. (A) Scheme illustrating in-cell ... Figure 6. eGFP-mRNA with azido-modified poly(A) tail can be labeled in living cells. (A) Scheme illustrating in-cell click reaction in mammalian cells transfected with ARCA-N<sub>3</sub>-eGFP-mRNA. (B) Confocal microscopy of HeLa cells transfected with eGFP-mRNA with azido modifications at the poly(A) tail (ARCA-N<sub>3</sub>) or with in vitro clicked poly(A) tail (ARCA-click). Sample with ARCA-N<sub>3</sub>-eGFP-mRNA was then subjected to DBCO-SRB treatment to achieve bioorthogonal fluorescence labeling in living cells, followed by washing steps. After incubation overnight, all cell samples were fixed and hybridized using Stellaris eGFP probe with Quasar 670 dye. SRB and Q670 fluorescence show mRNA labeling. Same cell samples were used as for translational analysis (Figure 5). Scale bars are 20 μm. 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 Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Nucleic Acids Res, Volume 47, Issue 7, 06 February 2019, Page e42, https://doi.org/10.1093/nar/gkz084 The content of this slide may be subject to copyright: please see the slide notes for details.