Expanding the chemical diversity of RNA by transcriptional incorporation of amino acid- and glycosyl-modified nucleotides

With the increasing interest in RNA-based therapies, there is a pressing need to incorporate new chemistries 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 investigate 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 modifications 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 ( View larger version (16K): org.highwire.dtl.DTLVardef@29b6daorg.highwire.dtl.DTLVardef@a3c425org.highwire.dtl.DTLVardef@1a27a4aorg.highwire.dtl.DTLVardef@ab0e9_HPS_FORMAT_FIGEXP M_FIG C_FIG