Polymerase Chain Transcription: Exponential Synthesis of RNA and Modified RNA

Abstract

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.

Enzymatic Synthesis, Amplification, and Application of DNA with a Functionalized Backbone

“…[26] As an initial exploration of the utility of SFM4-3, we demonstrated that when combined with thermocycling, it can exponentially produce large quantities of RNA or 2'-F RNAv ia ap rocess we refer to as polymerase chain transcription (PCT). [27] In addition, we used SFM4-3 to select 2'-F-modified aptamers and found that the modifications impart increased specificity. [28] While the modified nucleotides accessed by SFM4-3 impart the oligonucleotide products with interesting attributes,both the 2'-F and 2'-OMe moieties leave much functional potential to be desired.…”

Enzymatic synthesis of random sequences of RNA and RNA analogues by DNA polymerase theta mutants for the generation of aptamer libraries

“…It should be mentioned here that Romesberg and coll. ( 48 ) managed to develop an engineered DNA polymerase capable of amplifying directly RNA and modified RNA such as 2′ fluoro RNA. Also, these authors developed a third type of base pair that could be tried in the near future to be incorporated by CS13 mutant ( 49 ).…”

A guide to large-scale RNA sample preparation

“…To allow substrate tolerance of Thermococcus gorgonarius DNA polymerase, the group developed a mutant enzyme capable of synthesizing RNA and modified nucleic acids while retaining its stability toward heat denaturation [ 70 ]. The Romesberg group recently reported a variant of the Stoffel fragment of the Taq DNA polymerase (SFM4-3) [ 71 ] that was shown to exponentially amplify two different RNA fragments from one DNA template, primed by two asymmetric DNA oligonucleotides [ 5 ] (Fig. 2c ).…”