Figure 1.

Novel strategy for the enzymatic preparation of site‐specifically spin‐labeled long RNAs using an expanded genetic alphabet. Top: the full‐length double‐stranded DNA template is either generated A) by solid‐phase DNA synthesis of several short oligonucleotides followed by a six‐letter fusion PCR using unnatural nucleoside triphosphates (TPs) dTPT3 TP8 and dNaM TP8 in addition to the canonical nucleoside triphosphates or B) by six letter PCR using dTPT3 TP and dNaM TP amplifying from a plasmid template employing modified forward and reverse primers. Bottom: the novel nitroxyl‐modified nucleotide TPT3^NO TP (1) is incorporated into RNA through genetic alphabet expansion transcription.9