A combined in vitro/in vivo selection for polymerases with novel promoter specificities

The DNA-dependent RNA polymerase from T7 bacteriophage (T7 RNAP) has been extensively characterized, and like other phage RNA polymerases it is highly specific for its promoter. A combined in vitro/in vivo selection method has been developed for the evolution of T7 RNA polymerases with altered promoter specificities. Large (10(3)-10(6)) polymerase libraries were made and cloned downstream of variant promoters. Those polymerase variants that can recognize variant promoters self-amplify both themselves and their attendant mRNAs in vivo. Following RT / PCR amplification in vitro, the most numerous polymerase genes are preferentially cloned and carried into subsequent rounds of selection.

A T7 RNA polymerase library that was randomized at three positions was cloned adjacent to a T3-like promoter sequence, and a 'specialist' T7 RNA polymerase was identified. A library that was randomized at a different set of positions was cloned adjacent to a promoter library in which four positions had been randomized, and 'generalist' polymerases that could utilize a variety of T7 promoters were identified, including at least one polymerase with an apparently novel promoter specificity. This method may have applications for evolving other polymerase variants with novel phenotypes, such as the ability to incorporate modified nucleotides.