Abstract Title: In vitro Selection of Fast Catalytic Purine Nucleotide Synthase
Ribozymes.
Matthew W.L. Lau, Danny T.O. Shum and Peter J. Unrau
Simon Fraser University
RNA nucleotides are the basic building blocks for RNA. However, in modern
metabolism the synthesis of nucleotides is catalyzed by protein enzymes. Here we report the in
vitro selection of fast ribozymes capable of promoting the synthesis of a purine nucleotide (6thioguanosine monophosphate) from tethered 5-phosphoribosyl 1-pyrophosphate (pRpp) and 6thioguanine (6SGua) (1). The two most proficient purine synthases, called RA and MA, have
apparent efficiencies of 230 and 284 M-1min-1 respectively. Compared to previously selected
pyrimidine nucleotide synthase ribozymes, these ribozymes are 50-100 times more efficient
(2,3). Interestingly, RA has a Km of ~80 M, while MA has no detectable affinity for 6SGua,
indicating that considerably different forms of substrate recognition are utilized by the two
ribozymes. Both ribozymes showed good substrate discrimination against 6-thioguanosine
analogues, with only a slight tolerance for substitutions at the 2 position. It is of interest that the
protein enzyme HGPTase and a naturally occurring aptamer found in the xpt-pbuX mRNA also
discriminate weakly in a similar pattern.
Currently, we are focusing on determining the secondary structure of MA and a third
ribozyme isolate called MF (which contains sequence regions that can potentially fold into a
previously selected pyrimidine synthase ribozyme). Ribozyme isolates were lightly mutated and
then randomly recombined (4). Random recombination involves the digestion of ribozyme DNA,
followed by ligation to generate a diverse pool of ribozyme fragments that are linked in random
orientation and order. Sequences with structural regions that are of importance to purine
nucleotide synthesis will be selected for, while those missing such regions will be selected
against. The ability of ribozymes to synthesize both purine and pyrimidine nucleotides as
building blocks strongly suggests that RNA molecules could have preceded proteins in a
hypothetical RNA based metabolism.
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