Title: Characterizing Purine Nucleotide Synthase Ribozymes using Non-Homologous Recombination
M.W.L. Lau, D.T. Shum and P.J. Unrau,
Simon Fraser University, Burnaby, British Columbia, V5A 1S6, Canada
Speaker: Matt Lau

RNA nucleotides are the basic building blocks for RNA. In modern metabolism the synthesis of nucleotides is catalyzed by protein enzymes. One such protein catalyzed salvage pathway involves the reaction between 5-phosphoribosyl 1-pyrophosphate (pRpp) and a purine or pyrimidine base. Previously we demonstrated that pyrimidine and purine nucleotide synthase ribozymes could mimic these salvage pathways by using tethered pRpp reacting with either 4-thiouracil or 6-thioguanine (6SGua) [1,2]. Comparison between these two classes of ribozymes showed that the purine synthase ribozymes were on average 50-100 times more efficient than their pyrimidine counterparts [1,2].

In a continuation of this work, we seek to determine the secondary structures of purine nucleotide synthase ribozymes in order to compare with the previously characterized pyrimidine nucleotide synthases. To address this issue, we performed non-homologous recombination [3] and selection on two purine nucleotide synthase ribozyme isolates MA and MF. MA was chosen since it was the fastest out of all the sequences analyzed, while MF was interesting in that it had the potential to fold into a structure similar to that of a pyrimidine nucleotide synthase ribozyme. The recombined MA and MF pools were constructed such that each pool contained randomly recombined sequence elements relative to its parent. The two pools were selected for highly truncated sequences [3] that were able to promote 6SG synthesis. After six rounds of selection, we isolated short reactive species from both pools, and sequencing data revealed a large deletion region for both, removing ~50 nucleotides relative to each of the two 125 nucleotide long parent. We are currently working on deducing the secondary structure from the minimal core motifs of the isolates. This structural understanding should strengthen our knowledge of both small molecule RNA catalysis and the informational complexity required to produce catalytically active RNAs.

[1] P.J. Unrau and D.P. Bartel, Nature., 395, 260 (1998).

[2] M.W.L. Lau, K.E.C. Cadieux and P.J. Unrau, JACS., 126, 15686 (2004).

[3] Q.S. Wang and P.J. Unrau, RNA., 11, 404 (2005).