Structural characterization of an RNA polymerase ribozyme and functional improvement by in vitro encapsulated selection

At the heart of the “RNA World” hypothesis is the requirement for an RNA polymerase ribozyme capable of replicating itself and other RNA molecules. The most recent variant, isolated from multiple generations of in vitro selection, is the B6.61 RNA polymerase ribozyme that is capable of extending an RNA primer in a template-dependent fashion by 20 nucleotides. It contains a 5' ligase core domain that confers phosphodiester bond formation ability and a poorly understood 3' accessory domain that is proposed to attribute its polymerization ability. In my research presented in this thesis, we study the structure of the accessory domain of B6.61 by mutagenesis, chemical probing and crosslinking experiments and provide molecular constraints that are useful for modeling the three-dimensional structure of the ribozyme. Secondly, I have used in vitro encapsulated selection in an attempt to improve the overall processivity of B6.61.