Fig 1.
qPCR assay performance on plasmid DNA.
The tRNApyl and pylRSwt qPCR assays were tested with a tenfold serial dilution of their respective plasmids spanning 300–3×108 copies. Graphing the Ct versus the log of plasmid copies demonstrates a linear range of 300–3×108 copies for tRNApyl and 300–3×107 copies for pylRSwt. A simple linear regression shows R2 values approaching 1 and slope values close to -3.3.
Table 1.
Primers, probes, DNA oligo templates.
Fig 2.
qPCR assay performance on single stranded DNA oligos corresponding to their target cDNA.
(A) Sequence of DNA oligos with the qPCR primers in blue and the qPCR probes in red. 5’-3’ orientation is shown by the direction of the arrows. The position of the primers and probe for both mature and unprocessed tRNApyl are shown on the cloverleaf schematic of the tRNA. The primers for mature tRNApyl quantify total tRNApyl (mature and unprocessed). The reverse primer for unprocessed tRNApyl is specific for this species. (B) tRNApyl assay with the tRNApyl DNA oligo. (C) tRNApyl unprocessed assay with the tRNApyl unprocessed DNA oligo. (D) CHO-K1 tRNAmet assay with the CHO-K1 tRNAmet DNA oligo. (E) CHO-K1 tRNAphe assay with the CHO-K1 tRNAphe DNA oligo. The qPCR assays were tested using a tenfold serial dilution of their respective DNA oligo spanning 300–3×108 copies. Plotting the Ct versus the log input of DNA oligo copies show that all qPCR assays have a linear range of 300–3×108 with R2 values approaching 1 and slopes near -3.3.
Table 2.
Specificity of tRNApyl and tRNApyl unprocessed qPCR assays on single stranded DNA oligo.
Fig 3.
Quantifying RNA expression levels of unprocessed and mature tRNApyl in cell lines.
(A) A reporter mRNA construct was designed to assess amber suppression activity. The reporter encodes an RFP-GFP fusion interrupted by an amber stop codon. All transfected cells show RFP expression, but only amber suppression competent cells express the RFP-GFP fusion. (B) Six clones isolated from an amber suppression competent host were transfected with the RFP-GFP mRNA and exposed to nnAA. Cells were grown for 24 h. and fluorescence expression examined by flow cytometry. Six positive clones showing RFP and GFP signals were identified. A cell line showing no GFP expression is shown for comparison (Neg). (C) RNA from the six amber suppression competent cell lines (A-F) was examined by the tRNApyl and tRNApyl unprocessed assays to quantify the extent of tRNApyl processing. (D) A schematic representation of the workflow for isolating and analyzing nucleic acids from cell lines. The data show that mature tRNApyl constitutes >88% of the tRNApyl forms present in the samples and indicate efficient processing of the tRNApyl in CHO cells.
Fig 4.
Quantifying RNA expression levels of native CHO-K1 tRNAs, tRNApyl and pylRSwt in engineered cell lines.
RNA from six stable cell lines expressing pylRS/tRNApyl were assayed for endogenous tRNAphe, tRNAmet, as well as tRNApyl and pylRSwt. The data demonstrate that pylRSwt is expressed at comparable levels to the endogenous CHO-K1 tRNA. tRNApyl is expressed at ~1000-fold higher levels than endogenous tRNA.
Table 3.
Quantifying RNA expression levels of unprocessed and mature tRNApyl in cell lines.
Table 4.
Quantifying RNA expression levels of native CHO-K1 tRNAs, tRNApyl and pylRSwt in cell lines.
Fig 5.
CHO-K1 B2M assay performance on CHO-K1 genomic DNA.
The CHO-K1 B2M qPCR assay was tested against a fourfold serial dilution of CHO-K1 genomic DNA spanning 0.025–102.4 ng gDNA. Graphing the Ct versus the log of plasmid copies demonstrates a linear range of 0.025–102.4 ng gDNA with R2 values approaching 1 and slope values close to -3.3.
Table 5.
Quantifying gene copy number in genomic DNA.
Table 6.
Comparing the observed tRNApyl copy number to the predicted tRNApyl copy number.
Fig 6.
Comparing cell line DNA copy number to RNA expression for tRNApyl and pylRSwt assays.
Copy numbers of pylRS and tRNA were determined in six amber suppression competent cell lines. (A) The calculated DNA copy numbers plotted relative to the corresponding RNA expression levels. A loose correlation between copy number and expression is observed for pylRSwt; however, tRNA expression levels do not seem to depend on copy number. (B) The GFP:RFP ratios plotted relative to corresponding RNA expression levels.
Table 7.
Comparing cell line RNA expression of tRNApyl and pylRS to GFP:RFP ratio.