The Search for a Ribonucleotide Reductase in the "RNA World": In-Vitro Selection of Terminally Reduced 3'Deoxy-RNAs from Random RNA Libraries.


Dyna Shirasaki

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Our research explores the theory that life began in an "RNA World," where RNA alone was responsible for both carrying the genetic code as well as catalyzing reactions.We are searching for an RNA which will perform the reduction of its own 3' terminus into a deoxyribonucleotide (via an intramolecular reaction) when placed in the appropriate chemical environment.Initially, our focus has been developing a negative in-vitro selection assay which possesses the ability to remove RNA molecules from a pool containing a mixture of both RNA and 3' deoxy-RNA, leaving the 3' deoxy RNA untouched.In our experiments, a mixture containing 16mer 3' deoxy-RNA and RNA ranging from 20-90 nucleotides in length was treated with periodate, thereby oxidizing the cis-diols on the 3' terminus of the RNA molecule to aldehydes.The mixture was then exposed to agarose-based hydrazide beads, allowing the aldehyde functional groups on the ends of the RNA molecules to form hydrazone bonds with the beads.The beads were removed and the remaining pool was tested for the amounts of RNA and 3' deoxy RNA remaining. Various conditions were tested to determine optimal conditions.Currently, our best result involves a two-fold increase in the 3' deoxy-RNA population of the pool.


W. Reef Hardy




Norris Research Fellowship, Packard Foundation

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