Thermal cycling transcription boosts RNA production.

Efficient RNA production is essential for therapeutic applications. Currently, mRNA is generated by in vitro transcription with RNA polymerases at a constant temperature. The success of these transcriptions depends on various factors including sequence composition and size. Sometimes, especially for long RNAs, achieving efficient transcript yields are challenging. We developed a novel approach by incorporating a high-temperature denaturation step. Our protocol includes the following two steps of thermal cycling RNA transcription: the initial enzyme-driven RNA transcription step is done at RNA polymerase active temperature (37 {degrees}C); then the nucleic acids melting step is carried out at elevated temperatures (55-70{degrees}C); after the reaction is cooled to the temperature of optimal polymerase activity, the first step is repeated. Denaturation enables the generation of optimized, more stable mRNAs, which alleviates the mRNA instability that often occurs during RNA-based therapeutic development. For example, this new thermal cycling RNA transcription approach can employ high GC content, thus increasing RNA stability. For long and difficult templates, the combination of sequence optimization by LinearDesign algorithm in conjunction with transcription via thermal cycling significantly improved mRNA production. Thermal cycling transcription dramatically increased the efficiency of most RNA products while reducing costs, facilitating RNA-based therapeutic development.