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
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].
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  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  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.
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 M.W.L. Lau, K.E.C. Cadieux and P.J. Unrau, JACS., 126, 15686 (2004).
 Q.S. Wang and P.J. Unrau, RNA., 11, 404 (2005).