Valero, J.; Neis, K.; Civit, L.; Fjelstrup, S.; Gockert, M.; Kjems, J.

With the increasing interest in RNA-based therapies, there is a pressing need to incorporate new chemis-tries into more complex RNA molecules. These modifications can protect RNA from degradation, improve its pharmacokinetics, and enhance its targeting properties. Here we describe the enzymatic synthesis of chemically modified RNA derivatives using a mutant T7 RNA polymerase to incorporate 23 different base modifications alongside stabilizing ribose modifications, such as 2'-fluoro and 2'-deoxy groups. To investi-gate the impact on transcription efficiency and fidelity, we employed a pool of 38 template sequences and analyzed the transcripts by next-generation sequencing of the cDNA. Results demonstrated that all modi-fications were successfully incorporated into RNA, with transcription efficiency influenced by three main factors: type of modification, base modified, and the sequence context. Misincorporation levels during transcription and reverse transcription into cDNA were generally low (<1%) but included noticeable ex-ceptions for some nucleobase-modification combinations. As a robust proof-of-concept we demonstrat-ed the selection of Histidine-U modified aptamer, relying on multiple rounds of transcription and amplifi-cation, binding Influenza hemagglutinin protein with low nanomolar KD. We anticipate that this work will significantly contribute to the design and production of chemically modified RNAs with novel functionali-ties, advancing applications in biomedicine and synthetic biology.