Scheme 1.

mRNAs and chemo‐enzymatic modifications used in this study. A) 5′‐Clean Cap (5′‐CC) mRNA is produced via IVT. B,C) N ⁶‐Modification of adenosine at the transcription start nucleotide using CAPAM and AdoMet (S‐adenosyl‐l‐methionine) or AdoMet analogs yields methylated Clean Cap (5′‐CC‐N ⁶mA_m) or propargylated Clean Cap (5′‐CC‐N ⁶pA_m), respectively. D) Modification of the poly(A) tail of 5′‐CC‐mRNA using poly(A) polymerase and 2′‐azido ATP followed by labeling with Cy5 yields 5′‐CC‐3′‐Cy5‐mRNA. E) 5′‐ARCA (5′‐AC) mRNA is produced via IVT. F) N ²‐Modification of m⁷G using GlaTgs and AdoMet results in N ² methylated ARCA (5′‐N ²m‐AC)‐mRNA. G) Modification of the poly(A) tail of 5′‐AC‐mRNA using poly(A) polymerase and 2′‐azido ATP followed by labeling with Cy5 yields 5′‐AC‐3′‐Cy5‐mRNA.