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Cited by 129RNA and protein synthesis mechanismsRNA Research and SplicingRNA Interference and Gene Delivery

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Segmented poly(A) tails significantly reduce recombination of plasmid DNA without affecting mRNA translation efficiency or half-life

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Trepotec, Zeljka, Geiger, Johannes, Plank, Christian, Aneja, Manish K., Rudolph, Carsten·2019·RNA
DOI 10.1261/rna.069286.118PMC PMC6426288
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Abstract

Extensive research in the past decade has brought mRNA closer to the clinical realization of its therapeutic potential. One common structural feature for all cellular messenger RNAs is a poly(A) tail, which can either be brought in cotranscriptionally via the DNA template (plasmid- or PCR-based) or added to the mRNA in a post-transcriptional enzymatic process. Plasmids containing poly(A) regions recombine in E. coli, resulting in extensive shortening of the poly(A) tail. Using a segmented poly(A) approach, we could significantly reduce recombination of plasmids in E. coli without any negative effect on mRNA half-life and protein expression. This effect was independent of the coding sequence. A segmented poly(A) tail is characterized in that it consists of at least two A-containing elements, each defined as a nucleotide sequence consisting of 40–60 adenosines, separated by a spacer element of different length. Furthermore, reducing the spacer length between the poly(A) segments resulted in higher translation efficiencies compared to homogeneous poly(A) tail and reduced recombination (depending upon the choice of spacer nucleotide). Our results demonstrate the superior potential of segmented poly(A) tails compared to the conventionally used homogeneous poly(A) tails with respect to recombination of the plasmids and the resulting mRNA performance (half-life and translational efficiency).

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Figures

FIGURE 1. Schematic representation of all combinations of poly(A) modifications tested in the current study. Different homo- or heteropolymeric poly(A) stretches were inserted downstream from the gene of interest (GOI).
FIGURE 1. Schematic representation of all combinations of poly(A) modifications tested in the current study. Different homo- or heteropolymeric poly(A) stretches were inserted downstream from the gene of interest (GOI).CC BY-NC · PubMed Central
FIGURE 2. Quantification of poly(A) tail recombination for A120 and segmented poly(A) tails of poly(A)3 × 40_6 and poly(A)2 × 60_6. (n) Total number of clones of d2EGFP, luciferase, and hEPO sequences tested with a particular poly(A) format.
FIGURE 2. Quantification of poly(A) tail recombination for A120 and segmented poly(A) tails of poly(A)3 × 40_6 and poly(A)2 × 60_6. (n) Total number of clones of d2EGFP, luciferase, and hEPO sequences tested with a particular poly(A) format.CC BY-NC · PubMed Central
FIGURE 3. Determination of d2EGFP protein expression and mRNA quantification of different poly(A)-containing d2EGFP mRNAs post-transfection in A549 cells. (A) Mean MFI (median fluorescence intensity) at 4-, 24-, 48-, and 72-h post-transfection, measured by FACS in A549 cells. (B) d2EGFP mRNA quantification in A549 cells. (C) mRNA productivity was calculated by dividing the mean MFI (FACS data; A) by the mRNA amounts (real-time PCR data; B) and normalizing these ratios to those observed with poly(A)120 construct. Values represent mean ± SD of three replicates. Statistical significance was assessed by two-way ANOVA test with P-values: (*) P < 0.5, (**) P < 0.01, (***) P < 0.001, (****) P < 0.0001.
FIGURE 3. Determination of d2EGFP protein expression and mRNA quantification of different poly(A)-containing d2EGFP mRNAs post-transfection in A549 cells. (A) Mean MFI (median fluorescence intensity) at 4-, 24-, 48-, and 72-h post-transfection, measured by FACS in A549 cells. (B) d2EGFP mRNA quantification in A549 cells. (C) mRNA productivity was calculated by dividing the mean MFI (FACS data; A) by the mRNA amounts (real-time PCR data; B) and normalizing these ratios to those observed with poly(A)120 construct. Values represent mean ± SD of three replicates. Statistical significance was assessed by two-way ANOVA test with P-values: (*) P < 0.5, (**) P < 0.01, (***) P < 0.001, (****) P < 0.0001.CC BY-NC · PubMed Central
FIGURE 4. Determination of luciferase expression and mRNA quantification of different poly(A)-containing luciferase mRNA 24-h post-transfection in A549 cells. (A) Luciferase activity, measured as relative light units (RLU: arbitrary units) in protein lysates from A549 cells transfected with different poly(A)-containing luciferase RNA measured 24-h post-transfection. (B) Luciferase mRNA quantification in A549 cells. (C) mRNA productivity was calculated by dividing the luciferase expression values (RLU; A) by the mRNA amounts (real-time PCR data; B) and normalizing these ratios to those observed with poly(A)120 construct. Values represent mean ± SD of six replicates. Statistical significance was assessed by two-way ANOVA test with P-values: (*) P < 0.5, (**) P < 0.01, (***) P < 0.001, (****) P < 0.0001.
FIGURE 4. Determination of luciferase expression and mRNA quantification of different poly(A)-containing luciferase mRNA 24-h post-transfection in A549 cells. (A) Luciferase activity, measured as relative light units (RLU: arbitrary units) in protein lysates from A549 cells transfected with different poly(A)-containing luciferase RNA measured 24-h post-transfection. (B) Luciferase mRNA quantification in A549 cells. (C) mRNA productivity was calculated by dividing the luciferase expression values (RLU; A) by the mRNA amounts (real-time PCR data; B) and normalizing these ratios to those observed with poly(A)120 construct. Values represent mean ± SD of six replicates. Statistical significance was assessed by two-way ANOVA test with P-values: (*) P < 0.5, (**) P < 0.01, (***) P < 0.001, (****) P < 0.0001.CC BY-NC · PubMed Central
FIGURE 5. Quantification of secreted human erythropoietin protein levels as measured via ELISA in supernatants from HEK293 cells transfected either with poly(A)120- or poly(A)2×60-containing EPO mRNA at 24 h (A), 48 h (B), and 72 h (C) post-transfection. Values represent mean ± SD of three replicates. Statistical significance was assessed by two-way ANOVA test with P-values: (**) P < 0.01, (***) P < 0.001, n = 3.
FIGURE 5. Quantification of secreted human erythropoietin protein levels as measured via ELISA in supernatants from HEK293 cells transfected either with poly(A)120- or poly(A)2×60-containing EPO mRNA at 24 h (A), 48 h (B), and 72 h (C) post-transfection. Values represent mean ± SD of three replicates. Statistical significance was assessed by two-way ANOVA test with P-values: (**) P < 0.01, (***) P < 0.001, n = 3.CC BY-NC · PubMed Central
FIGURE 6. Relative quantification of CFTR protein in 16HBE14o- lysates as measured via western blot. (A) 16HBE14o- cells were transfected either with poly(A)120- or poly(A)2×60_6-containing hCFTR mRNA and protein lysates analyzed at 24 and 48 h post-transfection. (B) Densitometry analysis of western blot images. Values represent mean ± SD of two replicates. Statistical significance was assessed by paired t-test with P-values: ns = P > 0.5.
FIGURE 6. Relative quantification of CFTR protein in 16HBE14o- lysates as measured via western blot. (A) 16HBE14o- cells were transfected either with poly(A)120- or poly(A)2×60_6-containing hCFTR mRNA and protein lysates analyzed at 24 and 48 h post-transfection. (B) Densitometry analysis of western blot images. Values represent mean ± SD of two replicates. Statistical significance was assessed by paired t-test with P-values: ns = P > 0.5.CC BY-NC · PubMed Central
FIGURE 7. Determination of luciferase expression and mRNA quantification of different poly(A)-containing luciferase mRNA at 24 h post-transfection in A549 cells. (A) Luciferase activity, measured as relative light units (RLU: arbitrary units), in protein lysates from A549 cells transfected with different poly(A)-containing luciferase mRNA. (B) Luciferase mRNA quantification in A549 cells transfected with different poly(A)-containing luciferase mRNA. (C) mRNA productivity was calculated by dividing the luciferase expression values (RLU; A) by the mRNA amounts (real-time PCR data; B) and normalizing these ratios to those observed with poly(A)120 construct. Values represent mean ± SD of six replicates. Statistical significance was assessed by two-way ANOVA test with P-values: (*) P < 0.5, (**) P < 0.01, (****) P < 0.0001.
FIGURE 7. Determination of luciferase expression and mRNA quantification of different poly(A)-containing luciferase mRNA at 24 h post-transfection in A549 cells. (A) Luciferase activity, measured as relative light units (RLU: arbitrary units), in protein lysates from A549 cells transfected with different poly(A)-containing luciferase mRNA. (B) Luciferase mRNA quantification in A549 cells transfected with different poly(A)-containing luciferase mRNA. (C) mRNA productivity was calculated by dividing the luciferase expression values (RLU; A) by the mRNA amounts (real-time PCR data; B) and normalizing these ratios to those observed with poly(A)120 construct. Values represent mean ± SD of six replicates. Statistical significance was assessed by two-way ANOVA test with P-values: (*) P < 0.5, (**) P < 0.01, (****) P < 0.0001.CC BY-NC · PubMed Central
FIGURE 8. Determination of luciferase expression and mRNA quantification of different poly(A)-containing luciferase mRNA at 24 h post-transfection in A549 cells. (A) Luciferase activity, measured as relative light units (RLU: arbitrary units), in protein lysates from A549 cells transfected with different poly(A)-containing luciferase mRNA. (B) Luciferase mRNA quantification in A549 cells transfected with different poly(A) containing luciferase mRNA. (C) mRNA productivity was calculated by dividing the luciferase expression values (RLU; A) by the mRNA amounts (real-time PCR data; B) and normalizing these ratios to those observed with poly(A)120 construct. Values represent mean ± SD of six replicates. Statistical significance was assessed by two-way ANOVA test with P-values: (*) P < 0.5, (****) P < 0.0001.
FIGURE 8. Determination of luciferase expression and mRNA quantification of different poly(A)-containing luciferase mRNA at 24 h post-transfection in A549 cells. (A) Luciferase activity, measured as relative light units (RLU: arbitrary units), in protein lysates from A549 cells transfected with different poly(A)-containing luciferase mRNA. (B) Luciferase mRNA quantification in A549 cells transfected with different poly(A) containing luciferase mRNA. (C) mRNA productivity was calculated by dividing the luciferase expression values (RLU; A) by the mRNA amounts (real-time PCR data; B) and normalizing these ratios to those observed with poly(A)120 construct. Values represent mean ± SD of six replicates. Statistical significance was assessed by two-way ANOVA test with P-values: (*) P < 0.5, (****) P < 0.0001.CC BY-NC · PubMed Central
FIGURE 9. Quantification of poly(A) tail recombination rate for segmented poly(A) tails with a single nucleotide spacer. (n) Total number of clones of luciferase tested with a particular poly(A) format.
FIGURE 9. Quantification of poly(A) tail recombination rate for segmented poly(A) tails with a single nucleotide spacer. (n) Total number of clones of luciferase tested with a particular poly(A) format.CC BY-NC · PubMed Central

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  1. Voet D , Voet JG . 2011. Biochemistry. 4th ed. John Wiley & Sons, New York.

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Metadata

Authors
Trepotec, Z., Geiger, J., Plank, C., Aneja, M. K., & Rudolph, C.
Affiliations
  • Ethris (Germany) (DE)
  • TUM Klinikum (DE)
ORCID
Venue
RNA
Publisher
Cold Spring Harbor Laboratory
Published
2019-01-15
Volume
25
Issue
4
Pages
507-518
ISSN
  • 1355-8382 (ISSN-L)
  • 1469-9001
DOI
10.1261/rna.069286.118
PMC
PMC6426288
OpenAlex
W2910680408
Cited by
129
References
44
Citations by year
2026: 20 · 2025: 29 · 2024: 25 · 2023: 22 · 2022: 20 · 2021: 10 · 2020: 2 · 2019: 1
FWCI
2.73
Type
journal article
Language
en
Open access
Locations
  • RNA (publishedVersion)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by-nc)
  • PubMed Central (publishedVersion, cc-by-nc)
  • PubMed (publishedVersion)
  • mediaTUM (Technical University of Munich) (submittedVersion, other-oa)
  • mediaTUM – the media and publications repository of the Technical University Munich (Technical University Munich) (submittedVersion)
Funders
NIM Excellence Cluster
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA and protein synthesis mechanisms
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Research and Splicing
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
Keywords
poly(A) tail, plasmid recombination, Escherichia coli, mRNA half-life, translation efficiency, spacer length, mRNA therapeutics, plasmid DNA
MeSH terms
A549 Cells, Animals, Base Sequence, Escherichia coli, Genetic Engineering, Half-Life, Humans, Plasmids, Poly A, Recombination, Genetic, RNA, Messenger, Transfection, Protein Biosynthesis, HEK293 Cells

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