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.