A comprehensive study of ribozyme-mediated nucleotide and sugar chemistries

The ability of RNA catalysts (ribozymes) to synthesize nucleotides as basic building blocks for the assembly of larger RNA polymers is an important aspect of the 'RNA World' hypothesis. To examine the ability of RNAs to catalyze this chemistry, we performed an in vitro selection in my first project and successfully isolated ribozymes capable of synthesizing a purine nucleotide (6-thioguanosine, 6SG) from tethered 5-phosphoribosyl 1-pyrophosphate (PRPP) and 6-thioguanine (6SGua). Compared to the previously selected pyrimidine synthase ribozymes, these ribozymes are 50-100 times more efficient. In a continuation of this work, we next deduced the secondary structure of two purine synthase ribozymes by performing a re-selection on two truncated pools for reactivity with 6SGua. Interestingly we were able to isolate for functional sequences that were ~2.5 fold shorter than their full-length parental ribozymes, and with much simpler secondary structure relative to the pyrimidine nucleotide synthase ribozymes. The ability of RNAs to synthesize both purine and pyrimidine nucleotides strongly suggests that RNA could have preceded proteins in a hypothetical RNA based metabolism. While the ribozymes isolated from our selection were successful in catalyzing purine nucleotide synthesis, they do require a pre-activated sugar substrate. This is not highly plausible in the RNA world since PRPP readily undergoes hydrolysis. To examine this phenomenon, in our third project we implemented a selection using a RNA pool tethered to ribose-5-phosphate and selected them for reactivity with 6SGua. After six selection rounds, a single RNA sequence dominated the selection. Interestingly, this ribozyme can produce three different products: two when tethered with PR and a third product when tethered with PRPP, which was determined to be 6SG. Our results suggest that this highly versatile ribozyme is capable of recognizing two similar substrates for the synthesis of different products, and thus demonstrates the promiscuous potential of ribozymes upon encountering of an alternative substrate.