Generation of Chimeric DNA-RNA Structures using Engineered Replisomes in vivo

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PROJECT SUMMARY/ABSTRACT The evidence of ribose in ancient catalytic machinery such as the ribosome as well as a universal set of cofactors which are derived from ribonucleic acids (RNA) has led to the hypothesis that RNA was once the dominant biopolymer in life. The transition from an RNA to DNA world raises a multitude of questions regarding how life was able to support such a dramatic change in the most fundamental information storage molecules without compromising survival. Recent studies have shown that certain randomly mutagenized strains of E. coli are in fact capable of supporting surprisingly high amounts of RNA in their genome, however it remains unclear the exact biochemical mechanisms that allow this to occur, and further studies of these strains is challenging due to inherent instability of their genomes. In this project I will be developing a novel polymerase system which could potentially limit RNA incorporation to small regions of DNA such as a plasmid. Targeting RNA incorporation in this way will allow for the first time a systematic method to study the effects of high ribose content in genes, without compromising host genomes. The proposal outlined here will focus on strategies for engineering the DNA polymerase within the multienzyme viral genome replication machinery known as the T7 replisome. In Specific Aim I, I will outline a plan to study the effects of focused mutations within the active site of T7 DNA polymerase. This will include the development and implementation of 96-well based screening method for the rapid determination of DNA polymerases with the ability to incorporate RNA permissively. Specific Aim II details a protocol for the selection of RNA permissive DNA polymerases from much larger mutant pools. A key feature of this aim is the combination of phage-display which has been used for the selection of nucleotide promiscuity in vitro along with a complementation-based selection strategy which further selects for mutant polymerases that retain functionality in vivo. We hypothesize that a novel combination of these strategies can be used to solve the inherent limitation of phage display to in vitro reaction conditions which may not accurately reflect intracellular conditions. Finally, Specific Aim III details a new approach toward the selection of mutant polymerases with the ability to incorporate RNA into replicating plasmids within a bacterial host. This method will rely on the use of alkyne labeled ribonucleotides which will provide chemical handles for the enrichment of plasmids that encode polymerases with relaxed sugar specificity. Together this project will create progress toward an orthogonal polymerase system for the construction of chimeric DNA-RNA plasmids in vivo, a task which is currently impossible using known synthetic biology tools.