Synthesis of Long RNA with a Site-Specific Modification by Enzymatic Splint Ligation

Synthesis of RNA molecules that contain an internal site-specific modification is important for RNA research and therapeutics. While solid-state synthesis is attainable for such RNA in the range of 100 nucleotides (nts), it is currently impossible with kilobase (kb)-long RNA. Instead, long RNA with an internal modification is usually assembled in an enzymatic 3-part splint ligation to join a short RNA oligonucleotide, containing the site-specific modification, with both a left-arm and a right-arm long RNA that are synthesized by in vitro transcription. However, long RNAs have structural heterogeneity and those synthesized by in vitro transcription have 3-end sequence heterogeneity, which together substantially reduce the yield of 3-part splint ligation. Here we describe a method of 3-part splint ligation with an enhanced efficiency utilizing a ribozyme cleavage reaction to address the 3-end sequence heterogeneity and involving DNA disruptors proximal to the ligation sites to address the structural heterogeneity. The yields of the synthesized kb-long RNA are sufficiently high to afford purification to homogeneity for practical RNA research. We also verify the sequence accuracy at each ligation junction by nanopore sequencing.