Confirmation of mRNA- and mRNA/cDNA-peptide conjugates
We confirmed the presence of synthesized peptide on the mRNA- and mRNA/cDNA-peptide conjugates with two different methods. First, trypsin digestion (targeting Lys and Arg) was applied to the 10aa-random mRNA-tag product with and without translation and revealed on a urea-SDS PAGE. All peptides contain multiple Lys residues at the fixed C-terminus region (Supplementary Figure 1) adjacent to the DNA tag, hence the digested product size should become almost equivalent to the mRNA-tag. As a result, the upper band in the post-translation product disappeared after 30 minutes of incubation, indicating that the band was indeed corresponding to the mRNA-peptide conjugate product (Figure 2D). Alternatively, by using the mRNA-tag product derived from fixed FLAG-control sequence (Supplementary Figure 1), the presence of conjugated peptides before and after the cDNA synthesis was confirmed by western blot analysis (Figure 2E). FITC fluorescence signal from the membrane transferred samples and chemiluminescence signal via anti-FLAG-HRP antibody clearly overlap at the same position, indicating the formation of both peptide conjugates. Note that under the SDS-PAGE condition, the double-stranded mRNA/cDNA-peptide conjugate migrated towards the cathode faster than the single-stranded mRNA-peptide conjugate due to the doubled charge from the phosphate backbone.