Directed evolution of T7 RNA polymerase variants using an 'autogene'
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Natural enzymes, when used in biotechnological applications are often found not well suited for the tasks. Enzyme properties can now be improved by rational design or by directed evolution to produce useful biocatalysts. The work described in this thesis is mainly focused towards developing a directed evolution method to evolve RNA polymerases with novel properties, such as the ability to use modified nucleotides as substrates. A variety of modified nucleotides can be imagined that can impart unique characteristics to RNA molecules into which they are incorporated. Modified nucleotides improve the functionality of RNA, but more importantly, they increase the stability of RNA towards nucleases thus lending the RNA amenable to various biotechnological applications. Modified RNA transcripts will also find applications in the in vitro selection of aptamers and ribozymes. We have developed a directed evolution method for the isolation of RNA polymerase variants with altered promoter specificities and novel substrate specificities vii using a construct called an “autogene”. The DNA-dependent RNA polymerase from T7 bacteriophage (T7 RNA polymerase) is being used for the directed evolution studies. In short, a library of T7 RNA polymerase variants was made by randomizing the gene of T7 RNA polymerase at amino acid positions that are important for a desired activity (for e.g.: altered promoter recognition). This gene was then cloned downstream of a T7 promoter, generating a so-called autogene library. Following transformation to E.coli, those polymerase variants that best recognized their adjacent promoter self-amplified both their mRNAs and themselves in vivo. The variant mRNAs extracted from the population as a whole will be roughly represented according to the activities of their corresponding variant polymerases. Following reverse transcription and PCR amplification in vitro, the most abundant polymerase genes were carried into subsequent rounds of selection. The method allows large (103 -106 ) polymerase libraries to be efficiently searched for their promoter recognition ability and fidelity. Autogene selection was subsequently modified with a reporter gene and used to screen polymerase variants that can incorporate modified nucleotides into the RNA backbone. We have successfully evolved a novel T7 RNA polymerase variant that transcribes 2’-O-methyl RNA. Other selections can also be envisioned using the autogene system to discover new polymerases with novel abilities. The polymerases thus evolved were used to construct modified RNA libraries to be used in in vitro selection of modified ribozymes.