High-salt transcription from enzymatically gapped promoters nets higher yields and purity of transcribed RNAs

Abstract

T7 RNA polymerase is commonly used to synthesize large quantities of RNA for a wide variety of applications, from basic science to mRNA therapeutics. This in vitro system, while showing high fidelity in many ways, is also well known for producing longer than encoded RNA products, particularly under high-yield reaction conditions. Specifically, the resulting product pool is contaminated by an often disperse collection of longer cis-primed extension products. In addition to reducing yield via the conversion of correctly encoded RNA to longer products, self-primed extension generates partially double-stranded RNAs that can trigger the innate immune response. Extensive and low-yield purifications are then required to produce therapeutic RNA. Under high-yield conditions, accumulating concentrations of RNA effectively compete with promoter DNA for polymerase binding, driving self-primed extension at the expense of correct initiation. In the current work, we introduce a simple and novel modification in the DNA to strengthen promoter binding, shifting the balance back toward promoter-driven synthesis and so dramatically reducing self-primed extension. The result is higher yield of the encoded RNA at the outset and reduced need for extensive purifications. The approach can readily be applied to the synthesis of mRNA-length products under high-yield conditions.

Leveraging Active Learning to Establish Efficient In Vitro Transcription and Translation from Bacterial Chromosomal DNA

“…We speculate that the selection for lower concentrations of potassium glutamate is a shift of the reaction buffer toward more favorable conditions for T7 RNA polymerase activity and the synthesis of mRNAs. Noticeably, established protocols for in vitro transcription reactions based on T7 RNA polymerase use buffers that avoid potassium (and other monovalent cations) since high ionic strength reduces the polymerase affinity for its DNA template. , In standard IVTT reactions, potassium is included as it is fundamental for different processes, including ribosome stability, but its deleterious effect on transcription is marginal in systems doped with high concentrations of template DNA. In our situation, instead, the reaction conditions suggested by active learning seem to reduce potassium in order to improve transcription in a system with a low template DNA concentration.…”

Understanding the impact of in vitro transcription byproducts and contaminants

“…To overcome the loss in yield, the T7 RNA polymerase needs to be forced into close proximity to the T7 promoter or have an improved binding affinity to the T7 promoter. The latter can be achieved by a small manipulation of the DNA template ( Malagoda Pathiranage et al, 2023 ). By introducing a nick to the −4 position into the DNA template the binding affinity from the T7 to the promoter is enhanced.…”

DNA-terminus-dependent transcription by T7 RNA polymerase and its C-helix mutants

“…Previous efforts (2,(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32) have been made to eliminate the dsRNA byproducts in IVT, which is the major source of immunogenicity for mRNA therapeutics. However, these works have barely focused on the dsRNA generated by promoter-independent antisense transcription.…”

“…Certain modified nucleotides inhibit the generation of dsRNA byproducts in T7-RNAP-based IVT (15,21). Other efforts to reduce the production of dsRNA byproducts include applying high-salt transcription conditions with tight-binding promoter variants (22), co-tethering promoter DNA and T7 RNAP on magnetic beads (23), adding competing 3′-capture DNA (24) or chaotropic agents (2), and lowering the Mg 2+ concentration (15,21). In addition, post-transcriptional purification techniques such as reverse-phase high-pressure liquid chromatography (HPLC) (18,25) or cellulose chromatography (26) can also reduce dsRNA contaminants.…”