Near-cognate suppression of amber, opal and quadruplet codons competes with aminoacyl-tRNAPyl for genetic code expansion

► Nonsense suppression by natural amino acids hinders genetic code expansion. ► Higher levels of near-cognate suppression found in opal versus amber codons. ► E. coli BL21 and MG1655 display higher background suppression than TOP10. ► PylRS/tRNAUCAPyl cannot outcompete Trp incorporation at opal codons. ► PylRS/tRNAUCCUPyl is outcompeted by Arg-tRNAArg in quadruplet decoding.