Author: Tracey Ann Lincoln

Publisher: ProQuest

Published: 2009

Total Pages: 208

ISBN-13: 9781109124675

RNA enzymes based on the R3C ligase ribozyme motif were previously shown to undergo both self- and cross-catalytic replication, albeit very inefficiently. In an attempt to develop efficient cross-replicating RNA enzymes that can undergo self-sustained exponential amplification in the absence of proteins, in vitro evolution was carried out to optimize their catalytic activity. Two enzymes, E and E, ́ were selected for their ability to catalyze each other's synthesis from a total of four component substrates (A, B and A, ́ B)́. The optimized cross-replicating RNA enzymes were able to undergo self-sustained exponential amplification at a constant temperature in the absence of proteins or other biological materials. Amplification occurs with a doubling time of about one hour, and can be continued indefinitely, provided the system is replenished with substrates. Populations of various cross-replicating enzymes were constructed and allowed to compete for a common pool of substrates. This was demonstrated through serial transfer experiments which employed 5 [muM of each type of A, A, ́ B, and B ́and initiated with 0.1 [muM of each type of E and E.́ After 5 h incubation, 5% of the reaction mixture was removed and transferred to a separate reaction vessel that contained a fresh supply of substrates. This process was repeated for up to 20 transfers, during which recombinant replicators arose and grew to dominate the population. These replicating RNA enzymes can serve as an experimental model of a genetic system. Many such model systems could be constructed, allowing different selective outcomes to be related to the underlying properties of the genetic system.