In vitro selection of catalytic RNAs in emulsion


The RNA world hypothesis describes an early stage of life when RNA fulfilled both genomic and catalytic roles. One great effort to support this model is by generating catalytic RNAs (ribozymes) that can catalyze essential RNA world functions. One of these RNA world functions is establishing an energy metabolism in the form of chemically activated nucleotides. Trimetaphosphate has been shown to be a prebiotically plausible activating source that generates nucleoside triphosphates from nucleosides. A main goal of the Muller lab is to generate ribozymes that can establish an energy pathway by using trimetaphosphate. Such a ribozyme could utilize trimetaphosphate and nucleosides to generated chemically activated NTPs. NTPs have an essential role in modern life (as cofactors and monomers for genetic polymerization), and having a prebiotically plausible pathway for its generation would greatly support the RNA world hypothesis. Previous projects from the Muller lab have established a ribozyme that can triphosphorylate its own 5' end. Although this was significant in establishing that triphosphorylation can be catalyzed by ribozymes, it did not generate freely diffusing NTPs. Here I describe a selection for a nucleoside triphosphorylation ribozyme as well as additional RNA evolutions and engineering to accomplish this goal. The main efforts taken were the evolution of a polymerase ribozyme towards a thio-modified nucleotide and characterizing the emulsion environment necessary for the selection to work

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