Sprecher
Beschreibung
In the RNA world, improvements to RNA replication would permit more complex functional RNA molecules to evolve, which would in turn select for further improvements to replication in a bootstrapping process that presaged the emergence of complex cellular life and genetically encoded proteins. Only vestigial traces of the RNA world are left in modern life, but polymerase ribozymes have been evolved de novo in the laboratory. To improve these ribozymes’ activity and study RNA-based life in the laboratory, polymerases have been evolved in vitro to synthesize functional RNA molecules from RNA templates. As polymerase activity improves, the complexity of the functional RNA target can be increased, driving further improvements in polymerase activity in a bootstrapping process analogous to the evolution of genome complexity in the RNA world. Over more than sixty generations of in vitro evolution, polymerases have been selected to synthesize first simple ligand-binding RNA aptamers, then simple self-cleaving ribozymes, and most recently a 97-nucleotide ligase ribozyme that is related to the catalytic core of the polymerase itself and more than half its length. Over these generations, polymerases have accumulated more than 30 mutations and have undergone a major structural rearrangement of the catalytic core. Polymerases first improved efficiency by increasing rates of nucleotide incorporation by more than 300 fold and lowering the binding constant for primer-template duplexes from more than 3 mM to less than 1 µM. In the most recent generations, under selection to synthesize complex ribozymes that are more easily inactivated by mutation, polymerase accuracy has improved, enabling synthesis of 50-fold more unmutated RNA products from the most complex RNA templates. As polymerase activity improves further, it may soon be possible to extend selection toward polymerase synthesis of component pieces of itself. Once a polymerase achieves self-replication, long-term propagation of the polymerase and its autonomous Darwinian evolution would represent a reconstruction of RNA life, and enable the direct study of the RNA world in the laboratory.
