RNA therapeutics have emerged as a promising candidate for treating many serious diseases that do not respond to traditional medications, creating a greater need for faster and more cost-effective methods of synthesizing RNA in vitro. One of the greater challenges of synthesizing RNA in the lab is the tendency for impurities to arise as RNA is transcribed in solution, adding time and costs to the process through additional purification steps. Researchers from the University of Massachusetts Amherst have now designed a process that prevents impurities without hampering transcription, resulting in a higher yield of pure RNA in a shorter amount of time and at lower cost.
Double-stranded RNA impurities occur when T7 RNA polymerase rebinds to and extends encoded RNA, which is common when producing large amounts of RNA that accumulate in a solution. Time- and cost-intensive purification methods are required to remove these impurities, which also result in an overall loss of yield. The researchers sought to eliminate these impurities and generate pure RNA from the first step by increasing the salinity of the solution to prevent T7 RNA polymerase from rebinding to product RNA. To ensure that the high salt conditions did not also inhibit T7 RNA polymerase from binding to the promoter DNA, the researchers co-tethered the polymerase and DNA template to magnetic beads. This process enabled a high yield of RNA to be generated from the template while nearly eliminating the formation of double-stranded impurities.
Because the product RNA generated using this system is already highly pure, the method reduces the need for intensive purification processes. Preventing the formation of double-stranded impurities also prevents a loss in yield and greatly reduces the possibility of impurities being left in a final product. The tethered bead system can also be washed and reused, further increasing the potential yield of pure RNA. The team’s study was published in the Journal of Biological Chemistry.
“Rather than having to purify RNA, we’ve figured out how to make clean RNA right from the start,” said senior author Craig Martin. “Our method can be more than ten times better at producing pure RNA than current processes.”
The team hopes to further scale up the process by developing a “flow reactor” that would serve as a continuous pipeline for producing pure RNA.