Extended Data Fig. 3: Relationship between cDNA retrieved from bio-mREX and fluorescence obtained from in vivo genetic code expansion.
From: Adding α,α-disubstituted and β-linked monomers to the genetic code of an organism
Relationship between the acylation signal measured, by bio-mREX, for stmRNAs and the GFP fluorescence signal measured for intact, translation-competent tRNAs. For stmRNAs, active aminoacyl-tRNA synthetases (aaRS) lead to the acylation of their encoding stmRNAs, which by bio-mREX get extended, separated and ultimately reverse transcribed. This results in the cDNA of the active synthetase, which can be quantified by qPCR. In the case of an inactive aaRS the stmRNAs is not acylated and no cDNA is produced in bio-mREX experiments. Therefore, the activity of a synthetase in bio-mREX correlates with the number of cDNA molecules measured by qPCR. In canonical translation an active aaRS enzyme leads to an acylated, intact, cognate tRNACUA which is used in protein translation. Inactive aaRS enzymes lead to non-acylated tRNAs, which are not used in protein translation. The production of GFP protein from GFP(150TAG)His6, as measured by GFP fluorescence, reports on the acylation of tRNACUA, as well as the other steps in the production of protein.
