Incorporation of Pseudouridine into mRNA Yields Superior Nonimmunogenic Vector with Increased Translational Capacity and Biological Stability

• p. 2 Results, first section

  In wheat germ extracts, ~50% less

• p. 12 Figure 1b, wheat germ extract bars

  Wheat germ extract

Judge's note: Rendered Figure 1b from the PDF at 500 dpi. On the "Fold increase in translation" axis the wheat-germ Ψ bars read ~0.57 (blue, TEVlucA50) and ~0.28 (orange, capRen) against the 1.0 dashed reference line; the two "None" bars sit at 1.0 as expected. The legend (p012) confirms the normalisation: "Fold increase in translation was calculated by normalizing the measured relative light units to those obtained with nonmodified mRNA". So the single figure "~50% less" covers two reductions of 43% and 72% (mean ~57%). The direction and the qualitative point (Ψ gives less protein in wheat germ) are correct and the tilde signals approximation, so this is loose rounding over two constructs rather than a wrong value — downgraded from the reviewer's PROBABLE to a minor wording imprecision.

Second check: another model agreed

• p. 17 Figure 4, panel b key and legend (b)

  Animals were killed at 2 and 4 hours postinjection

• p. 9 Materials and Methods, "Administering mRNA to mice"

  Mice were killed when indicated (1, 4, 6, or 24 hours)

• p. 17 Figure 4b legend

  Animals were killed at 2 and 4 hours

• p. 4 Results, in vivo section

  readily detectable at 15 minutes, 1 hour, and 4 hours postinjection (Figure

Only the authors can say what is right here.

Judge's note: Opened adjudicate/crops/C025-a.png and C025-b.png, plus the full page image extract/png/main-p017.png. The Figure 4b plot key on the page image plainly shows two bars labelled "2 h" (open) and "4 h" (grey), matching the legend text, while the Methods parenthesis enumerates 1, 4, 6 or 24 hours; every other in vivo timepoint in the paper (Fig 4c/4d: 1, 4, 24 h; Fig 4e/4f: 6 h; plasmid: 24 h) falls inside that list, so 2 h is the sole undocumented time. I searched the Results and Methods text for an explanation and the Results give no time for the organ-distribution panel ("thus in subsequent studies only spleens were examined"), so nothing in the article reconciles the two. Rated S3 rather than S1 because the discrepancy is an unresolved inconsistency over one timepoint and only the authors can say whether the Methods list is incomplete or the panel label should read 1 h. The sub-point that the text's "readily detectable at 15 minutes, 1 hour, and 4 hours postinjection" (crop C025-d) exceeds the 1/4/24 h lanes of Fig 4d is not confirmed: that sentence co-cites Supplementary Figure S6, which is not among the files and is the plausible location of the 15-minute data. The sub-point comparing the Fig 4b legend with panel c is a different experiment (spleen time course) and is not a defect.

Second check: another model agreed

• p. 3 Results, cultured cells

  In all of cell types, the Ψ modification

Judge's note: Read the sentence on the p003 page image. The printed text is "In all of cell types, the Ψ modification conferred the highest translational yield to RNA and,". Confirmed on the image, so not a text-layer artefact. Fix: "In all cell types" or "In all of the cell types". Cosmetic only; meaning is unaffected.

Second check: none (judge only)

• p. 3 Results

  in both number of positive cell and intensity

• p. 3 Results

  translational capacity of all tested RNA

Judge's note: Read both quoted sentences on the p003 page image. "in both number of positive cell and intensity" is confirmed as printed and is a clear number-agreement error. The cluster's second item, "significantly improved translational capacity of all tested RNA (caplacZ, caplacZ-An, and capGFP-An)", is also as printed, but "RNA" as a mass noun is defensible English, so I have narrowed the statement to the "positive cell" error, which is the part that is unambiguously wrong. Cosmetic only.

Second check: none (judge only)

• p. 3 Results

  had unproportionally higher luciferase level

Judge's note: Read the sentence on the p003 page image; the printed word really is "unproportionally", not a text-layer artefact. The paper itself uses the standard adverb elsewhere (p005: "resulted in proportionally greater production of luciferase"), so the usage is internally inconsistent as well as non-standard. Meaning is recoverable, so cosmetic.

Second check: none (judge only)

• p. 4

  when exposed to RNA containing 5′-triphoshate; however, replacement of

• p. 4 Results, "Superior translation of Ψ-containing mRNA is independent of RIG-I", first paragraph

  when exposed to RNA containing 5′-triphoshate

• p. 4 Results, same paragraph, two lines later

  contained a 5′-triphoshate

• p. 4 RIG-I section, para 1

  RNA containing 5′-triphoshate

Judge's note: Read both occurrences on the p004 page image and on the C007-b/C007-c crops at high magnification; the second "p" really is absent in both places, so this is not a dropped glyph in the text layer. The same page spells the word correctly two sentences later ("Transcripts containing either 5′-triphosphates (ppplucA50)") and again in "the triphosphates were replaced with cap", and the Figure 3 legend on p016 prints "had triphosphate at its 5′-end", so the misspelling is inconsistent within the paper. No variant spelling exists. Cosmetic.

Second check: another model agreed

• p. 4

  Efficient translation of Ψ-containing mrnA in mice

• p. 16 Figure 3, title

  Superior translation of Ψ-modified mrnA is independent of retinoic acid-inducible protein I (RIG-I)

• p. 16 Figure 3 title

  Superior translation of Ψ-modified mrnA is independent

Judge's note: Opened adjudicate/crops/C009-a.png (p. 4) and C009-b.png (p. 16 Figure 3 title). Both rendered images show lowercase m, r, n followed by capital A — "mrnA" — not a small-caps or extraction artefact; the surrounding body text on the same crops renders "mRNA" correctly in the same font. A grep of extract/main.txt finds exactly these two occurrences of "mrnA" in the article. Purely cosmetic capitalisation.

Second check: another model agreed

• p. 4 RIG-I section

  derived from RIGI(−/−) and wild-type mice

• p. 16 Figure 3 legend

  Wild-type (wt) and RIGI(−/−) mouse embryonic fibroblasts

• p. 4 RIG-I section

  RIGI(−/−) and wild-type mice

• p. 16 Figure 3 legend

  RIGI(−/−) mouse embryonic fibroblasts

Judge's note: Confirmed both on the page images (p004, p016) and the crops; the hyphen is genuinely absent, so not a dash-rendering artefact. The correct form appears nearby in the same paper: p004 "did not induce IFN-β from the RIG-I(−/−) MEFs", p006 "using RIG-I(−/−) MEFs", the Methods "Culture medium collected from RIG-I(−/−)", and the Figure 3 panel axis labels themselves read "RIG-I wt" and "RIG-I −/−". A purely typographic inconsistency, so cosmetic rather than a label error that would mislead about the population.

Second check: none (judge only)

• p. 5 Discussion, first paragraph

  in vitro–transcribed mRNAs have never been vigorously tested for in vivo gene replacement

Judge's note: Read the sentence on the p005 page image: "Unlike viral- and plasmid-based vectors, in vitro–transcribed mRNAs have never been vigorously tested for in vivo gene replacement, only for therapeutic vaccination (reviewed in ref. 14)." The contrast drawn is with vectors that have been thoroughly evaluated, so "rigorously" is the intended word; "vigorously" (with force or energy) does not carry that sense. A wrong-word substitution whose meaning is still recoverable, hence cosmetic.

Second check: none (judge only)

• p. 6 Discussion, second paragraph

  activated by structural motifs present in mRNA-containing uridine

Judge's note: Page 6 image shows "mRNA-containing uridine17 but not Ψ modification" printed within one line, with a real hyphen. The compound is reversed; the next sentence contrasts nonmodified and Ψ-modified mRNAs, so "uridine-containing mRNA" is meant. Meaning is recoverable, so cosmetic.

Second check: none (judge only)

• p. 6 Discussion

  but not, Ψ modified, mRNA

Judge's note: Page 6 image shows "unmodified, but not Ψ modified, mRNA" with no hyphen; the same page prints "Ψ-modified mRNAs" and "Ψ-modified RNAs" with one. Hyphenation inconsistency only; in predicate-like position the unhyphenated form is defensible.

Second check: none (judge only)

• p. 8 Assembling the RNA complex

  Fifty microliter of such a complex

Judge's note: Page 8 image shows "Fifty microliter of such a complex was used". Singular unit after a plural number; cosmetic.

Second check: none (judge only)

• p. 9

  8-week-old, weighting 18–23 g were prewarmed using

• p. 9 Administering mRNA to mice

  weighting 18–23 g

Judge's note: Page 9 image shows "weighting 18–23 g". Wrong word for "weighing"; cosmetic.

Second check: another model agreed

• p. 17 Figure 4 legend, opening sentence

  In vitro-transcribed capTEVlucA50 (1,866 nt) with or without Ψ modifications were extended

• p. 17 Figure 4, panel a (gel image, labelled "a")

  capTEVlucA50

• p. 17 Figure 4 legend start

  In vitro-transcribed capTEVlucA50 (1,866 nt) with or without Ψ modifications

Judge's note: Page 17 image: the legend runs straight from the title into the description of the gel in panel a with no "(a)"; later panels are labelled (b), (c,d), (d), (e), (f). The Figure 2 and Figure 3 legends on pages 14 and 16 do print "(a)". Cosmetic omission.

Second check: none (judge only)