Polymerase Chain Transcription: Exponential Synthesis of RNA and Modified RNA

Journal of the American Chemical Society

5 years ago

Polymerase Chain Transcription: Exponential Synthesis of RNA and Modified RNA

Tingjian Chen, Floyd E. Romesberg

There is increasing demand for RNA and modified RNA oligonucleotides, but in contrast to DNA oligonucleotides, they are typically prohibitively expensive to chemically synthesize, and unlike longer RNAs, they are only inefficiently produced by in vitro transcription, especially when modified. To address these challenges, we previously reported the evolution of a thermostable DNA polymerase, SFM4-3, that more efficiently accepts substrates with 2′-substituents. We now show that SFM4-3 efficiently transcribes RNA or 2′-F-modified RNA and that it also efficiently PCR amplifies oligonucleotides of mixed RNA and DNA composition. In addition, with thermocycling and the use of a novel DNA template, we demonstrate a polymerase chain transcription (PCT) reaction that results in the exponential production of orders of magnitude more RNA or modified RNA than is available by conventional transcription. PCT is more efficient and general than conventional transcription and can produce large amounts of any RNA or modified RNA oligonucleotide at a fraction of the cost of chemical synthesis.

Publisher URL: http://dx.doi.org/10.1021/jacs.7b03981

DOI: 10.1021/jacs.7b03981