With this award, the Chemistry of Life Processes Program is supporting the research of Professor Donald Burke of the University of Missouri toward the development of RNA enzymes that catalyze phosphoryl transfer with peptide substrates. Phosphoryl transfer onto the 2'- and 5'-hydroxyls of RNA and DNA is a well-established catalytic capability of nucleic acids. In contrast, there have been no reports of ribozymes that add phosphates to, or remove phosphates from, other substrates, in spite of strong experimental proof-of-principle and sound chemical foundations arguing that such activity should be within reach of ribozyme catalysis. The research plan utilizes an innovative combination of new and established technologies, while building on strong preliminary studies of kinase ribozymes and aptamer-protein interactions. Identification and characterization of a ribozyme that manipulates the phosphorylation state of a peptide would represent a major advance in nucleic acid chemistry and chemical biology. The project further seeks to define the RNA's contribution to both substrate binding and catalysis, and to establish the roles of individual bases/sugars on the RNA in key mechanistic steps, particularly those involving proton transfer.
The project has potentially broad scientific impacts, as it seeks to push the limits of nucleic acid catalysis. Expanding RNA catalysis into the realm of peptide substrates would also deepen our understanding of ribozyme-peptide co-evolution during the origins of biological systems. Nucleic acid-based kinases and phosphatases also have potential longer term impacts in chemical and cell biology, particularly in the unraveling and manipulation of phosphorylation-based signaling pathways. This work will also impact the public through newspaper and online publications and via a collaborative project with MU School of Journalism aimed at strengthening communication at the intersection between Life Sciences and communication with the public. Finally, in this project, the PI seeks to train next generation scientists, through the mentoring of both undergraduate and graduate coworkers at the chemistry-biology interface.
DNA and RNA are both built from strings of "building blocks" (such as A, C, G and T), and they both fold into elegant 3-dimensional structures. The details of those structures determine how a given "string" can be used by cells or in biotechnology applications. For example, it is well known that some RNA strands fold into three-dimensional shapes that act as catalysts to accelerate chemical reactions, analogous to the protein enzymes that are essential to all living things. It is often argued that RNA's catalytic ability makes it a great candidate as the dominant catalyst during the earliest evolution of life. No one knows exactly what specific chemical processes led to the first organisms, or whether RNA would have been able to catalyze them. Our lab has long been interested in establishing what is really possible for RNA to do. Discovering New RNA Catalysts • Using a technology dubbed "evolution-in-a-tube," we identified many RNA molecules that catalyze a chemical reaction that is similar to one of the most important in biology. Specifically, these RNA molecules can "tag" other molecules, known as "acceptors," by attaching a phosphate onto them. • In the past decade, the cost and availability of DNA sequencing technology has plummeted to a point where it is readily accessible to any moderately funded researcher. However, the sheer amount of data can be hard to digest. A single experiment can produce the equivalent of the total information content of all the pixels in the Lord of the Rings trilogy in high-definition. The available software tools are either not well suited for experiments that rely on identifying a single sequence of DNA among millions of similar sequences, or they require the experimenter to have substantial abilities in writing computer code. In this study, we remedy this lack of software with "FASTAptamer." This user-friendly toolkit performs the early-stage analysis that that is critical to a wide variety of selection-based biotechnologies. How Do They Work? • In a recent study, we establish that the acceptor for one particular RNA is surprisingly similar to one seen in biology. In particular, the "acceptor" for this catalyst is a guanine nucleobase, making this a reaction that has never been observed before for RNA, although modern protein enzymes perform a similar reaction during the production of the building blocks of RNA. This is important because it reveals new ways in which RNA can catalyze reactions that are relevant both to modern biology the early evolution of life. • In another study, we identified key features of the chemical strategies that this RNA uses to carry out its reaction. By monitoring how the catalyst responds to changes in reaction conditions, we established that it uses a strategy known as "general base catalysis," in which it removes a proton from an as-yet-unknown molecular component, and that it takes advantage of the chemical properties of copper ions to make the phosphate more reactive. How Can We Use These New Catalysts? • We recently engineered this same RNA catalysts into an "RNA regulator." Specifically, we programmed it to add an extra phosphate onto other RNA molecules and demonstrated that the presence of the extra phosphate intered with the normal function of those RNAs. The field of Synthetic Biology combines biology and engineering to build useful biological systems. A long-term goal of this aspect of our work is to develop new tools for artificially regulating genes.
