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

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 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 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 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 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 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 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 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 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.