A practical approach to generate suitable de novo synthesis RNA template for a flavivirus RNA-dependent RNA polymerase

Due to its high transcription efficiency, bacteriophage T7 RNA polymerase (T7 RNAP) has long been utilized in bacterial and in vitro systems to generate large quantity of RNA for various purposes ( Studier and Moffatt, 1986). However, heterogeneity at the 3′-end of the RNA transcript remains a major limitation for in vitro RNA preparation using T7 RNAP. When approaching the end of its DNA template, T7 RNAP tends to add a few extra nucleotides not directed by the template strand sequence, resulting in a mixture of RNA products that are equal to or longer than the desired length ( Milligan et al., 1987). Various strategies have been used to possibly overcome this limitation, such as introducing 2′-methoxy groups to the last two nucleotides at the 5′-terminus of the DNA template strand ( Kao et al., 1999), seeking other high-efficiency RNA polymerase ( Zhu et al., 2014), using a general purpose RNA-cleaving DNA enzyme ( Santoro and Joyce, 1997), or introducing a self-cleaving...