In vitro selection of ligase ribozymes

Self-replication by RNA plays a central role in the RNA world theory of the origin of life. This thesis investigates the potential of RNA to catalyze a reaction related to RNA self-replication--RNA ligation.; Chapter 2 describes an attempt to use in vitro selection to improve the intermolecular ligation rate of a self-copying ribozyme derived from the sunY group I intron. When tethered to its partial P1 substrate helix by a long polynucleotide linker, the rate of self-ligation by the group I ribozyme approximates that of the intermolecular ligation. Variants of the ribozyme were selected for their ability to efficiently ligate an oligoribonucleotide to the tethered partial P1 substrate stem-loop. Eight rounds of in vitro selection from a pool of 10{dollar}sp{lcub}15{rcub}{dollar} independent variants of the ribozyme resulted in isolates that were only moderately more efficient at catalyzing intermolecular ligation than the starting ribozyme. The affinity of the ribozyme for the RNA substrate helix was largely unaffected by the mutations selected in vitro.; Chapters 3 and 4 report the in vitro selection and directed evolution of novel ligase ribozymes. The isolated ligase ribozymes catalyze the self-ligation of an oligoribonucleotide to their AMP-activated 5{dollar}spprime{dollar}-ends, in a reaction similar to that of protein DNA and RNA ligases. Eight different ligase ribozymes were isolated after four rounds of iterative selection from a pool of 10{dollar}sp{lcub}15{rcub}{dollar} RNAs containing both random sequence and a mutagenized ATP aptamer region. An additional six rounds of selection involving error-prone PCR allowed for the isolation of improved catalysts and one new ribozyme sequence. The ribozymes self-ligate at rates up to 0.4 hr{dollar}sp{lcub}-1{rcub}{dollar}, a rate 5 {dollar}times{dollar} 10{dollar}sp5{dollar} times higher than the templated background ligation rate, and at least three of the ribozymes catalyze the formation of 3{dollar}spprime{dollar}-5{dollar}spprime{dollar}-phosphodiester bonds. The ribozymes of the major sequence family found in the round 10 pool form a highly complex secondary structure consisting of ten stems incorporated within two long-range, nested pseudoknots. AMP-activation has proven to be a viable alternative to pyrophosphate activation for ribozyme-catalyzed ligation, thereby suggesting its possible role in analogous RNA world reactions.