The fastidious behavior of T7 RNA polymerase limits the incorporation of synthetic nucleosides into RNA transcripts, particularly at or near the promoter. The practically exclusive use of GTP for transcription initiation further compounds this challenge, and reactions with GTP analogs, where the heterocyclic nucleus has been altered, have not, to our knowledge, been demonstrated. The enzymatic incorporation of (th)GTP, a newly synthesized isomorphic fluorescent nucleotide with a thieno[3,4-d]pyrimidine core, is explored. The modified nucleotide can initiate and maintain transcription reactions, leading to the formation of fully modified and highly emissive RNA transcripts with (th)G replacing all guanosine residues. Short and long modified transcripts are synthesized in comparable yields to their natural counterparts. To assess proper folding and function, transcripts were used to assemble a hammerhead ribozyme with all permutations of natural and modified enzyme and substrate strands. The (th)G modified substrate was effectively cleaved by the natural RNA enzyme, demonstrating the isomorphic features of the nucleoside and its ability to replace G residues while retaining proper folding. In contrast, the (th)G modified enzyme showed little cleavage ability, suggesting the modifications likely disrupted the catalytic center, illustrating the significance of the Hoogsteen face in mediating appropriate contacts. Importantly, the ribozyme cleavage reaction of the emissive fluorescent transcripts could be followed in real time by fluorescence spectroscopy. Beyond their utility as fluorescent probes in biophysical and discovery assays, the results reported point to the potential utility of such isomorphic nucleosides in probing specific mechanistic questions in RNA catalysis and RNA structural analysis.