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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 (6-thioguanosine
monophosphate) from tethered 5-phosphoribosyl 1-pyrophosphate (pRpp) and 6-thioguanine
(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.
M.W.L. Lau, K.E.C. Cadieux and P.J. Unrau. Isolation of fast purine nucleotide synthase ribozymes. Journal of the American Chemical Society
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