Single-subunit RNA polymerases, KpnP, Ro45Iw, and CD23823, with precise terminal synthesis

T7 RNA polymerase (RNAP) is a highly active single-peptide polymerase that is widely used for in vitro RNA synthesis. T7 RNAP synthesizes RNA with a heterogeneous 3'-end, which sometimes causes problems. To date, some RNAPs (e.g., KP34 and Syn5) that synthesize RNA with more precise 3'-ends have been reported. However, they have their own characteristics and thus do not fully substitute T7 RNAP. To increase the number of usable RNAP repertoires, we searched for other RNAPs and their promoters in the phage RNAP database. From the first screening of nine RNAPs, we selected three RNAPs, named KpnP, Ro45Iw, and CD23823, together with their promoter sequences. Characterization of recombinant RNAPs revealed that compared to T7, these three polymerases exhibited similar RNA synthesis activities but preferred a slightly lower temperature. Additionally, CD23823 exhibited a slightly higher salt tolerance. Deep sequencing of the 5'- and 3'-termini of the synthesized RNA revealed that the three RNAPs, particularly CD23823, produced more homogeneous termini than T7 RNAP. These results indicate that these previously uncharacterized RNAPs, especially CD23823, are useful for RNA synthesis that requires precise 5'- and 3'-end sequences.