We have developed a range of T7 RNA polymerases, containing carefully selected mutations that control levels of by-products, such as dsRNA, and avoid complicated purification.
Based on thousands of RNA productions, we know which enzyme to select to get the best outcome, whether you are developing a vaccine or a therapeutic.
Contaminating dsRNA represents a serious limitation in downstream applications, as it impairs mRNA expression and can induce an undesired immune response. This is typically mitigated using downstream purification strategies, alteration of the IVT conditions, and the use of bacteriophage RNA polymerases homologs of T7 RNAP or its mutants. However, these strategies present problems of costly scaling, incomplete reduction of dsRNA contamination, or/and low yield of the target RNA.
Our patented T7RNAP produces both low levels of dsRNA (as low as 0.01% w/w), while at the same time maintaining the RNA yield.
• Enzymes tuned to your mRNA sequence
• Controlled immune activation for Vx
• Ultra-low dsRNA for Tx
• No need for dsRNA purification
Figure 1: The performance of RiboPerfectTM low dsRNA was compared with seven leading competitors that offer a low dsRNA T7RNAP variant. Fig 1a: Heat map showing RNA yield. Blue indicates the highest yield. White indicates the lowest yield. For each T7 RNAP variant (rows), a set of five different DNA templates of varying sizes (columns) was tested according to the manufacturer’s instructions. Yields of purified RNA were measured by UV spectrophotometry at 260 nm. Fig 1b: Heat map showing dsRNA content in purified RNA samples. White indicates the highest dsRNA content (≥ 0.3). Blue indicates the lowest dsRNA content. The content of dsRNA is represented for each T7 RNAP variant (rows), for a set of five different DNA templates (columns). The percentage of dsRNA was calculated by sandwich ELISA using specific mouse J2 and K2 monoclonal antibodies. RNA size: SecNLuc (0.78 kb), Fanca (4.55 kb), HER2 (3.8 kb), Fluc (1.81 kb), SUMO (1.02 kb), VEEV (8.6 kb).
Figure 2: Six selected RiboPerfectTM modulated dsRNA variants were used to perform IVT reactions using Fluc and SecNLuc as DNA templates (Fig. 2a and b, respectively). Yields of purified RNA for each variant were measured by UV spectrophotometry at 260 nm (Blue, left y-axis). The content of dsRNA was calculated by sandwich ELISA using specific mouse J2 and K2 monoclonal antibodies (white, right y-axis).