A Simple Approach to Improving RNA Synthesis: Salt Inhibition of RNA Rebinding Coupled With Strengthening Promoter Binding by a Targeted Gap in the DNA
Kithmie MalagodaPathiranage and Craig T. Martin, Methods in Enzymology, in press, 2023
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T7 RNA polymerase is widely used to synthesize RNA of any length, and long-standing protocols exist to efficiently generate large amounts of RNA. Such synthesis, however, is often plagued by so-called “nontemplated additions” at the 3’ end, which are in fact templated by the RNA itself and which give rise to double stranded RNA impurities in RNA therapeutics. These additions are generated by RNA polymerase re-binding to the product RNA (independent of DNA) and this rebinding is in competition with promoter binding. This chapter reports on a general approach that simultaneously weakens RNA rebinding by increasing salt, while at the same time increases promoter binding through manipulating the promoter DNA structure, shifting the balance away from self-primed extension. We present two approaches for use in different regimes. For (short) RNAs using synthetic oligonucleotides as DNA, promoter binding is strengthened by using a partially single stranded promoter construct already in wide use in the field. For the synthesis of RNA (of any length) from plasmid DNA, one can replicate the behavior of the first approach by introducing a targeted gap in the promoter, using a PCR primer containing an engineered deoxyuracil that is then excised by a commercially available enzyme system, to leave a promoter-strengthening gap. Both approaches are simple to implement, with only slight variations on standard synthesis approaches, making them valuable tools for a wide range of applications, from basic science to mRNA, CRISPR, lncRNA, and other therapeutics.