Uracil base modification improves transgene insertion efficiency in RNA templates with suboptimal 5′ modules. (A) Analysis of Rz-28(28) cleavage from a PP7 hp leader when either U or the substituted U analog is used for Rz synthesis. Reaction products were separated by 12% Urea-PAGE. Here and subsequently, altered mobility of the cleaved PP7 hp with different U analogs is due to folding and not differences in molecular weight (see Materials and Methods). (B) Quantification of insertion efficiency of SL-28 (green) and Rz-28(28) (gray) RNA templates when U or indicated U analogs are used for RNA template synthesis, as determined by flow cytometry. Values indicate mean ± SD, n = 3. (C) PCR amplification of 3′ (top) and 5′ (bottom) rDNA-transgene junctions from gDNA in a representative replicate of the experiment in B. (D) Analysis of HDV gRz cleavage from a PP7 hp leader when either U or the substituted U analog is used for Rz synthesis. Reaction products were separated by 12% Urea-PAGE. (E) Quantification of insertion efficiency of various 5′ module RNA templates synthesized with either U (green) or fully substituted with pseudouridine (gray), as determined by flow cytometry. Average fold enhancement, defined as the percentage of cells expressing GFP yielded by a 5′ module template synthesized with pseudouridine over the same template synthesized with U, is indicated below each 5′ module. Values indicate mean ± SD, n = 3. (F) PCR amplification of 3′ (top) and 5′ (bottom) rDNA-transgene junctions from gDNA in a representative replicate of the experiment in E. (G) Average transgene copy number per cell as measured within the 3′ module (blue) or 5′ promoter region (dark gray) relative to RPP30 by ddPCR. ddPCR was performed using gDNA extracted from cells PRINTed with U or pseudouridine-substituted RNA templates. Values indicate mean ± SD, n = 3. (H) Percentage of productive insertions (PI%, the copy number of promoter relative to 3′ module) as determined from ddPCR analysis in G. Fold enhancement, defined as the percentage of productive insertions yielded by a 5′ module template synthesized with pseudouridine over the same template synthesized with U, is indicated below each 5′ module. Values indicate mean ± SD, n = 3.
