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. 2020 Mar;26(3):345–360. doi: 10.1261/rna.073858.119

FIGURE 3.

FIGURE 3.

High-temperature IVT with TsT7-1 leads to a reduction in 3′-extended by-products. (A) Denaturing gel analysis of IVT products synthesized from short IVT template-1 using TsT7-1 performed at a temperature range of 37°C to 55°C. Chemically synthesized short IVT template 1 RNA was used as a control. Equal amounts were loaded in each lane. (B) Intact mass spectrometry analysis of Template IVT short-1 and Template IVT short-2 using TsT7-1 under standard conditions (all four rNTPs; +rUTP or rCTP) or lacking one rNTP (−rUTP or −rCTP) performed at either 37°C or 50°C. Expected run-off transcript (including n + 1 and n + 2 products) is highlighted in orange, and products longer than the run-off transcript are highlighted in gray. (C) dsRNA immunoblot with J2 antibody and gel electrophoresis analysis of CLuc RNA synthesized with TsT7-1 or wild-type RNAP at a temperature range from 37°C to 55°C. For the immunoblots with TsT7-1, 1.0 µg of RNA was loaded for each mRNA. For the gel electrophoresis analyses with TsT7-1, 0.05 µg of RNA was loaded in each well. (D) Intact mass spectrometry analysis of IVT reactions in the presence or absence of TsT7-1 on chemically synthesized RNA (n, n + 1, n + 2) performed at either 37°C or 50°C. Products longer than the run-off transcript are highlighted in gray.