Role of a free-ending poly(A) tail on translation efficiency. (A) Rationale for using a type IIS restriction enzyme (eg, Sap1) instead of a type II restriction enzyme (eg, Spe1) for linearization of the plasmid template before in vitro transcription. Type IIS restriction enzymes cut adjacent to rather than within their recognition site and prevent an overhang of nucleotides derived from the vector backbone and remaining as 3′ attachment at the poly(A) tail. (B) Impact of an unmasked free poly(A) tail on translational efficiency in dendritic cells. Immature dendritic cells were electroporated with eGFP-A(67) RNA, which contains an unmasked poly(A) tail or with eGFP-A(67)ACUAG, in which 4 additional nucleotides are attached 3′ to the poly(A) tail. Electroporation without RNA or with RNase-digested RNA served as controls. Cells were harvested at different time points (3 hours, 6 hours, 24 hours, 48 hours, 72 hours, 120 hours, 168 hours, 192 hours), and the eGFP fluorescence of viable cells was measured by flow cytometry.