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Evolving thermostable reverse transcriptase activity in a DNA polymerase scaffold.

DNA polymerases are involved in all DNA synthesis that occurs in nature. Furthermore, DNA polymerases are the workhorses in numerous important molecular biological core technologies like the widely applied polymerase chain reaction (PCR), cDNA cloning, genome sequencing, and diagnostics based on nucleic acids. In each of these processes the DNA polymerase requires a DNA strand as a template for directing DNA synthesis. In contrast, RNA templates are processed with marginal activity. Neverthe less, it has been reported that some DNA polymerases like Thermus thermophilus (Tth) exhibit increased reverse tran scriptase (RT) activity exclusively in the presence of Mn ions. However, for many applications in mRNA diagnosis, for example, in pathogen detection or gene expression analysis, employment of Mn is not suited. Thus, we asked whether one is able to generate a DNA polymerase that is proficient to first reverse transcribe RNA into DNA and subsequently amplify the DNA by PCR without the requirement of Mn ions. Through this approach, new insights into DNA polymerase substrate recognition and valuable tools for applications should be gained. Methods of directed evolution have been shown to be promising to engineer nucleic acid polymerases with altered properties. Alterations have mainly been achieved by directed molecular evolution by using genetic complementa tion and/or screening, phage display, or in vitro compart mentalization. Herein, we show that the generation of a new DNA polymerase function, that is, RT PCR activity, is achievable through iterative screening of small libraries of DNA polymerases. Our example is one of the rare examples where using an arbitrary randomized (nonfocused) library was sufficient to identify a new DNA polymerase activity. To evolve reverse transcriptase activity in an N terminal, shortened form of DNA polymerase from Thermus aquaticus (Klentaq) we randomized the open reading frame by error prone PCR (epPCR). Protein expression was conducted in 96 well plates, and enzymes were screened for PCR activity