polya tail by  (TriLink)

Journal: Nucleic Acids Research

Article Title: PolyA tail segmentation improves the stability of the template DNA and increases the translatability of in vitro transcribed mRNA

doi: 10.1093/nar/gkaf1412

Figure Lengend Snippet: Identity confirmation and stability assessment of modified polyA sequences at the DNA and RNA levels. (A) Comparison of polyA tail sequence instability levels in plasmid DNA vectors encoding FLuc mRNA with various polyA variants across two bacterial strains: TOP10 and NEB ® Stable Competent E. coli . Instability levels were expressed as the percentage of clones with altered polyA tail length or sequence relative to the original construct. (B) Chromatographic profiles of mRNA with modified polyA sequences. Absorbance at 260 nm shows similar elution patterns across polyA constructs, confirming that sequence variations and spacer modifications do not affect mRNA synthesis. (C) Agarose gel electrophoresis of purified mRNA. (D) Confirmation of polyA structures at the DNA and RNA level. Comparison of plasmid DNA and IVT RNA sequencing for constructs R1, R6, S4, and S8. For clarity, only data for polyA fragments are shown. DNA sequencing was performed by the Sanger method; raw data shows individual nucleotide reads in the polyA region of a plasmid DNA construct encoding FLuc. Color coding: green- adenine, red- thymine, blue- cytosine, black- guanine. RNA sequencing was performed using the Nanopore DRS method for mRNAs obtained from templates generated by linearization of the plasmid DNA. The results are expressed as changes in current [pA] as a function of sequencing time [s]. Each significant change in the current indicates the occurrence of a heteronucleotide relative to adenine.

Article Snippet: Overall, using NEB ® Stable Competent E. coli reduced polyA tail loss by ~2–3-fold during amplification (Fig. ).

Techniques: Modification, Comparison, Sequencing, Plasmid Preparation, Clone Assay, Construct, Agarose Gel Electrophoresis, Purification, RNA Sequencing, DNA Sequencing, Generated

Journal: Nucleic Acids Research

Article Title: PolyA tail segmentation improves the stability of the template DNA and increases the translatability of in vitro transcribed mRNA

doi: 10.1093/nar/gkaf1412

Figure Lengend Snippet: The composition of the polyA tail affects the expression analysis of the encoded FLuc. (A–D) Time-dependent production of FLuc in cultured cells transfected with mRNAs carrying various polyA tails. Graphs show relative activity (chemiluminescence) of FLuc (which is proportional to luciferase protein levels) in cells following transfection with different mRNA variants, including all references (R1–R6) as well as mRNAs with modified 3′-tail sequences (S1–S8). Relative activity of FLuc was measured in four cell lines: A549 ( A ), HepG2 ( B ), HEK293T ( C ), and JAWSII ( D ) at four time points post-transfection (4, 16, 24, and 48 h). (E–H) Overall FLuc production in different cell lines for each mRNA variant relative to reference mRNA (R1) represented as the AUC (area under the curve). Graphs show the total FLuc protein from all time points normalized to the same data for R1. ( E ) The total FLuc protein expression in the A549 cell line with statistical significance relative to R1 (A90). ( F ) The total FLuc protein expression in HEK293T cell line with statistical significance relative to R1 (A90). ( G ) The total FLuc protein expression in the HepG2 cell line with statistical significance relative to R1 (A90). ( H ) The total FLuc protein expression in the JAWS II cell line with statistical significance relative to R1 (A90). Statistical significance was assessed using the Kruskal–Wallis test with Dun’s multiple comparisons test (medians) for HepG2 and HEK293T, (ns,* P < .05,** P < .01), and using one-way ANOVA (mean ± SD) for A549 and JAWSII (* P < .05, ** P < .01, *** P < .001, **** P < .0001).

Article Snippet: Overall, using NEB ® Stable Competent E. coli reduced polyA tail loss by ~2–3-fold during amplification (Fig. ).

Techniques: Expressing, Cell Culture, Transfection, Activity Assay, Luciferase, Modification, Variant Assay

Journal: Nucleic Acids Research

Article Title: PolyA tail segmentation improves the stability of the template DNA and increases the translatability of in vitro transcribed mRNA

doi: 10.1093/nar/gkaf1412

Figure Lengend Snippet: Analysis of mRNAs with polyA tail variants encoding mKate2_PEST: expression kinetics and stability. (A) Time-dependent expression profiles of mKate2_PEST in A549 and HEK293T cells transfected with mRNAs carrying different modified 3′ terminal sequences. Protein expression levels are presented as the sum of raw fluorescence intensities measured over multiple time points (5–77 h). (B) Correlation between mRNA half-life and translation efficiency in HEK293T and A549 cells. The graph depicts the half-life of mRNAs encoding mKate2_PEST ( X -axis) in relation to the protein synthesis efficiency per mRNA molecule ( Y -axis). Each point corresponds to a specific 3′-tail sequence variant, with circles representing HEK293T cells and triangles indicating A549 cells. Dashed lines indicate the half-life of mKate2_PEST in HEK293T (179 min) and A549 (259 min) cells. All values are presented as means ± standard deviation (SD). (C) Total mKate2_PEST expression levels in A549 and HEK293T cells, represented as the AUC (area under the curve). AUC values were calculated as the sum of the areas under the fluorescence intensity curves, providing a comparative measure of overall protein production for each mRNA variant. Statistical significance was assessed using one-way ANOVA (* P < .05, ** P < .01, *** P < .001, **** P < .0001).

Article Snippet: Overall, using NEB ® Stable Competent E. coli reduced polyA tail loss by ~2–3-fold during amplification (Fig. ).

Techniques: Expressing, Transfection, Modification, Fluorescence, Sequencing, Variant Assay, Standard Deviation

Journal: Nucleic Acids Research

Article Title: PolyA tail segmentation improves the stability of the template DNA and increases the translatability of in vitro transcribed mRNA

doi: 10.1093/nar/gkaf1412

Figure Lengend Snippet: Overall FLuc production in different cell lines for the polyA modifications with multiple cytosine spacers and extended length. (A–D) Time-course analysis of FLuc expression in cell lines: A549 ( A ), HEK 293T ( B ), HepG2 ( C ), and JAWSII ( D ). The graphs illustrate FLuc protein expression for polyA tail modifications (variants: S9, S10, S11, S12, S13, S14, and S15) compared to mRNAs carrying the canonical polyA sequences R1 and R5, as well as the best variant from SET I - S8. (E–H) Area under the curve (AUC) analysis of FLuc protein production over time for each polyA tail modification in A549 ( E ), HEK 293T ( F ), HepG2 ( G ), and JAWSII ( H ) cell lines. AUC values were calculated by summing the areas under the curves (A–D) at each time point. All values are presented as mean ± standard deviation (SD). Statistical significance was assessed using one-way ANOVA (* P < .05, ** P < .01, *** P < .001, **** P < .0001).

Article Snippet: Overall, using NEB ® Stable Competent E. coli reduced polyA tail loss by ~2–3-fold during amplification (Fig. ).

Techniques: Expressing, Variant Assay, Modification, Standard Deviation

Journal: Nucleic Acids Research

Article Title: PolyA tail segmentation improves the stability of the template DNA and increases the translatability of in vitro transcribed mRNA

doi: 10.1093/nar/gkaf1412

Figure Lengend Snippet: Extended segmented polyA tail (S 14 ) further enhances in vivo protein production from synthetic mRNA in mice. ( A ) Quantification of bioluminescence over time (4, 8, 12, and 24 h) following intravenous (i.v.) administration of FLuc-encoding mRNAs (10 µg/mouse) formulated in SM-102 LNPs. ( B ) Total bioluminescence signals calculated as the sum of bioluminescences measured at 4, 8, 12, and 24 h. All values are presented as mean ± standard deviation (SD). ( C – E ) Individual bioluminescence profiles of mice inoculated with FLuc mRNA variants. ( F ) Representative bioluminescence images taken 4 h after mRNA administration. ( G ) Concentrations of human alpha-1 antitrypsin (hA1AT) in the sera of mice inoculated i.v. with 1 µg of mRNA encoding hA1AT, formulated in SM-102 LNPs. All data are presented as mean ± standard deviation (SD), with n = 5 mice per group. Statistical significance was assessed using one-way ANOVA (*** P < .001).

Article Snippet: Overall, using NEB ® Stable Competent E. coli reduced polyA tail loss by ~2–3-fold during amplification (Fig. ).

Techniques: In Vivo, Standard Deviation