A convenient method to generate and maintain poly(A)-encoding DNA sequences required for in vitro transcription of mRNA

Abstract

Generating mRNA in vitro to encode therapeutic or cell-modifying proteins is rapidly gaining favor. An important factor that determines efficiency of translation from in vitro transcribed mRNA is the length of the 3′ poly(A) sequence. However, reproducibly generating and maintaining templates from circular plasmids to have consistent lengths of the homo poly(A) sequences is challenging. The procedure reported here entails repeated restriction digestion with type IIS enzymes, ligation and circular plasmid propagation. The homopolymeric sequence of approximately 100 bp that is generated using the method is approximately equal to the number of 3′ A residues found in the mRNA of mammalian cells. Evaluating expression in vivo of a reporter transcript produced using this method showed efficient expression in vivo. METHODS SUMMARY A method that uses type IIS restriction enzymes to build a poly(A:T) tract of an mRNA-encoding sequence within a circular plasmid is described. Repeated asymmetric cleavage followed by ligation enables lengthening of the homo tract to an approximate maximum of 100 poly(A:T) base pairs. mRNA transcribed from this template is efficiently translated in vivo.