| Products/Services Used | Details | Operation |
|---|---|---|
| Gene Services | 5′ -6-FAM fluorescently tagged DNA fragments (Genscript, se- quences are shown in Supplementary Table S1) with or with- out T7 promoter were annealed in 10 mM Tris–HCl (pH 7.5) and 5 mM NaCl. Then, 10 nM DNA hairpins were incubated with various concentrations (0, 25, 50, 100, 200, 400 and 800 nM) of WT T7 RNAP or its G47W or E45K mutant in 40 mM Tris–HCl (pH 8.0), 15 mM MgCl2 , 2 mM spermidine and 5 mM DTT at 37◦C for 20 min. | Get A Quote |
The remarkable success of messenger RNA (mRNA)-based vaccines has underscored their potential as a novel biotechnology platform for vaccine development and therapeutic protein delivery. However, the single-subunit RNA polymerase from bacteriophage T7 widely used for in vitro transcription is well known to generate double-stranded RNA (dsRNA) by-products that strongly stimulate the mammalian innate immune response. The dsRNA was reported to be originated from self-templated RNA extension or promoter-independent transcription. Here, we identified that the primary source of the full-length dsRNA during in vitro transcription is the DNA-terminus-initiated transcription by T7 RNA polymerase. Guanosines or cytosines ... More
