Engineering the thumb loop of T7 RNA polymerase improves the integrity of long RNA transcripts

Incomplete transcripts complicate therapeutic mRNA manufacturing, particularly for long constructs such as self-amplifying RNA (saRNA). Their mechanisms remain poorly understood, hindering targeted improvements to mRNA integrity, a critical quality attribute. Here, our goal was to engineer the thumb loop of T7 RNA polymerase (RNAP) to improve transcript integrity. Using templates derived from a common 12 kb RNA sequence, we observed decreasing integrity as transcript length increased. Substitution of proline residues within the thumb loop improved integrity across 4, 8, and 12 kb transcripts, with several variants outperforming the previously reported K389A variant on the 12 kb template. Further substitutions identified M362K as the top performer and K363R as beneficial, implicating local conformation and electrostatic interactions. M362K retained its integrity-enhancing effect with N1-methylpseudouridine or 5-methylcytidine incorporated during transcription. These findings identify the thumb loop as a target for improving the integrity of long, chemically modified RNA transcripts.