123Fig. 2 In vivo nonsense suppression. a The non-canonical amino acid ( ncAA) is chemically cou-
pled to the dinucleotide phosphodesoxy-cytosine phospho-adenosine (pdCpA) via ester bond for-
mation between the free hydroxyl group (3′) of the ribose in the phospho-adenosine molecule and
the carboxyl group of the ncAA. The starting product of the ncAA is a cyanomethyl ester ( CME).
A protecting group ( PG) is shielding the alpha amino group from reactivity. b The ncAA-pdCpA
conjugate is subsequently ligated to a refolded tRNA that lacks the 3′ dinucleotide CA. Here, the
final product of the ligation is shown: a misacylated THG73 tRNA ( Tetrahymena thermaphila
glutamyl-tRNA U73G) carrying the anti-codon for TAG (CUA). c Efficiency of the ligation can be
estimated via denaturing TBE/Urea polyacrylamide (15 %) gel electrophoresis: the ligated THG73
(75-mer) is separated from the non-reacted 73-mer. RNA was visualized with a commercial nucle-
otide dye. d Qualitative proof of tRNA misacylation with the ncAA of interest can be achieved
by mass spectrometry analysis. Spectrum shows fragments of misacylated tRNA after digest with
S1 nuclease detected in reflector negative mode on a MALDI TOF instrument (CHCA matrix).
Numbers correspond to measured mass that are in agreement with theoretical calculations. pA,
5′-phosphoryl-adenosine; pA-AA-PG, 5′-phosphoryl-adenosine esterified with an amino acid ( AA)
that carries a protecting group ( PG) on the alpha amine (here: pA-Phe-NVOC)
Incorporation of Non-Canonical Amino Acids