Optimize in vitro transcription reactions: A modified T7 RNA Polymerase with reduced dsRNA formation

JENA BIOSCIENCE NEWS

Double-stranded RNA (dsRNA) is a critical by-product of wildtype T7 RNA Polymerase-based in vitro transcription. It needs to be minimized in final RNA preparations to reduce the risk of undesired immune responses[1-3]. Removal of such dsRNA impurities is typically performed by time-consuming & multi-step chromatographic processes[3-4].

HighYield T7 PURE RNA Synthesis Kit - based on a modified T7 RNA Polymerase (T7 PURE) – facilitates efficient RNA synthesis with reduced dsRNA formation (Fig. 1) thereby greatly streamlining down-stream purification protocols. Yields are comparable to wildtype T7 RNA Polymerase Mix after 2 h incubation at 37 °C (Tab. 1).

The kit is therefore ideally suited for RNA preparations where the dsRNA impurity level is critical for down-stream applications e.g. mRNA preparation for cell culture experiments.

Figure 1: HighYield T7 PURE RNA Polymerase Mix reduces dsRNA formation during in vitro transcription (IVT) compared to wildtype HighYield T7 RNA Polymerase Mix.

Table 1: Overview of available HighYield RNA Polymerase Kits

Product	Features	Yield after 30 min at 37 °C*	Yield after 2 h at 37 °C*
HighYield T7 RNA Synthesis Kit	Based on wildtype T7 RNA Polymerase Traditional variant	140 -160 µg	140 -160 µg
HighYield T7 P&L RNA Synthesis Kit	Based on T7 P&L RNA Polymerase Associated with decreased abortive transcription[5], increased 5‘ homogeneity[6] and increased 5‘ incorporation efficiency of GTP analogs[7]	140 -160 µg	140 -160 µg
HighYield T7 PURE RNA Synthesis Kit	Based on T7 PURE RNA Polymerase Reduced dsRNA formation compared to wildtype T7 and T7 P&L RNA Polymerase	100 -120 µg	140 -160 µg
HighYield T7 Enzyme Testkit	Contains HighYield T7, HighYield T7 P&L and HighYield T7 PURE RNA Poymerase Mix Designed to find the optimal RNA Polymerase Mix for a specific application

* reaction conditions: 1 µg DNA template (1400 nt RNA transcript), 7.5 mM each unmodified NTPs, 1x HighYield T7 Reaction Buffer, 2 µl each HighYield T7 WT, T7 PURE or T7 P&L RNA Polymerase Mix. Incubation for 30 min and 2 h at 37 °C. Read out: Fluorescence microplate assay.

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