Isolation of Fast Purine Nucleotide Synthase Ribozymes

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Abstract
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). The two most proficient purine synthases have apparent efficiencies of 284 and 230 M-1 min-1 and are both significantly more efficient than pyrimidine nucleotide synthase ribozymes selected previously by a similar approach. Interestingly, while both ribozymes showed good substrate discrimination, one ribozyme had no detectable affinity for 6-thioguanine while the second had a Km of ∼80 μM, indicating that these ribozymes use considerably different modes of substrate recognition. The purine synthases were isolated after 10 rounds of selection from two high-diversity RNA pools. The first pool contained a long random sequence region. The second pool contained random sequence elements interspersed with the mutagenized helical elements of a previously characterized 4-thiouridine synthase ribozyme. While nearly all of the ribozymes isolated from this biased pool population appeared to have benefited from utilizing one of the progenitor's helical elements, little evidence for more complicated secondary structure preservation was evident. The discovery of purine synthases, in addition to pyrimidine synthases, demonstrates the potential for nucleotide synthesis in an ‘RNA World' and provides a context from which to study small molecule RNA catalysis.

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