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Ongoing Projects

Currently, our laboratory focuses on exploring the chemical versatility of catalytic RNAs (ribozymes). The ability of RNA to recognize small substrates and promote difficult chemistry is poorly understood but is required to provide a solid foundation for the RNA world hypothesis. Specifically, we have been examining the ability of RNA to replicate itself, without the help of protein. Previous work has focused on this problem using in vitro selection (where ribozymes are isolated from large pools of random sequences by iterative rounds of selection and amplification). Dr. Unrau's important initial work involved selecting and evolving ribozymes able to form a nucleotide from a free pyrimidine base and a tethered form of activated ribose (PMID: 9751052). This ability to demonstrate RNA-mediated nucleotide synthesis provides strong support for the prior existence an RNA world (by suggesting a source of nucleotides for polymerization). These nucleotide monomers would have been the basic substrates for an RNA-templated RNA polymerase responsible for the replication of a primitive ribo-organism's genome. We continue to expand on this work in several key areas:

  • In vitro selection of ribozymes able to perform novel small molecule reactions. Examples include nucleotide synthesis, RNA polymerization and RNA capping. How might these ribozymes be engineered to work completely in trans and perform multiple turnover chemistry? We are actively exploring the chemical mechanism of these ribozymes.
  • Selection of ribozymes able to capture chemical energy. How could an ancient metabolism have been powered? One possibility, exploited by modern metabolism, is the oxidation of aldehydes to capture energy in the form of acyl-phosphate compounds. Can a ribozyme sequence be isolated that performs similar chemistry? Finding such a ribozyme would demonstrate that RNA-based life could have harvested both reducing power and activated carbon for metabolism, using a realistic abiotic source.
  • Development of new in vitro selection technologies. How can current in vitro selection methods be improved? A successful in vitro selection must isolate catalytic sequences from a huge background of inactive sequences. In practice, a substrate for the ribozyme reaction is tethered to the 3' or 5' end of the RNA pool molecules. Active sequences able to react with a second tagged substrate can be purified away from the inactive molecules in the pool and enriched. This approach has two drawbacks. First, tethering one of the substrates to the ribozyme makes it difficult to engineer true multiple turnover catalysts. Second, requiring the active site of the ribozyme to form at either the 3' or 5' end of the nucleic acid sequence places a heavy combinatorial constraint on the number of active sequences available in a random sequence pool. We are using a new encapsulated methodology to select for ribozymes completely in trans.
  • RNA in biology. We are generally interested in the regulatory role of RNA in biological systems and are interested in bacterial and eukaryotic systems of regulation. We are currently interested in bacterial regulatory RNAs such as the 6S RNA and eukaryotic small RNAs.