Noncoding RNA and Nucleic Acids Engineering
Ronald Breaker, Ph.D.

Ronald Breaker, Ph.D.

Henry Ford II Professor, Department of Molecular, Cellular and Developmental Biology
Professor, Molecular Biophysics and Biochemistry
Investigator, Howard Hughes Medical Institute
Room: KBT 506
Phone: 432-9389/432-6554
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B.S. University of Wisconsin-Stevens Point 1987; Ph.D. Purdue University 1992

Breaker LabOngoing investigations into the mechanisms of cellular life are revealing that sophisticated noncoding RNAs are involved in many key biochemical processes including information transfer, catalysis, signal transduction and molecular recognition. Breaker laboratory members are exploring the structural and functional diversity of RNAs that exist in modern cells, and are using test tube evolution methods to create RNAs and DNAs with entirely new functions. Several topics of special interest are currently being pursued that involve a range of techniques in the areas of nucleic acids biochemistry, microbiology and bioinformatics.

Riboswitches are regulatory domains usually embedded in messenger RNAs that sense specific metabolites or other signals and trigger changes in gene expression. We have established a robust program to discover and analyze metabolite–sensing riboswitches and have already uncovered dozens of distinct classes in bacteria. Validated examples are known to bind compounds fundamental to all life forms, such as coenzymes, nucleotide derivatives, or amino acids. Some riboswitches occur in tandem arrangements that provide sophisticated functions such as digital gene control and multi-input logic gate function. Importantly, we have shown that some riboswitches are targets for antibacterial compounds, which offers new opportunities for antibiotics drug discovery.

Large Noncoding RNAs
We are conducting bioinformatics searches to uncover highly–conserved RNAs, and these efforts are revealing many hundreds of different noncoding RNAs in bacteria and other organisms. In rare instances, we find unusually large and complex noncoding RNAs whose functions remain mysterious. Some are linked to virus replication or life under extreme conditions, while others are involved in gene regulation. Recently, we identified a large RNA chimera that self-splices in response to two chemical signals, which represents the most complex all–RNA device discovered to date. Our laboratory will continue to search for more examples of these large functional RNAs and establish their biochemical functions.

Nucleic Acids Engineering
We are also probing the structural and catalytic repertoire of nucleic acids by using test tube evolution. This method is modeled after the process of Darwinian evolution and is composed of iterative cycles of selection and amplification at the molecular level. We are using test tube evolution to isolate rare RNAs or DNAs with new and improved functions from trillions of variant or random-sequence molecules. For example, we have engineered RNA-based molecular switches that undergo activation or deactivation in the presence of small organic compounds, heavy metals, or even light. Even DNA can be forced to act as an enzyme, and we have created numerous examples of deoxyribozymes that catalyze chemical transformations. We are continuing to define the catalytic potential of both RNA and DNA under physiological conditions and to explore the range of chemical reactions catalyzed by novel nucleic acid enzymes when they are created outside the confines of cells.

Nucleic Acids and Early Evolution
The diverse tasks performed by natural noncoding RNAs perform in modern cells and by those we can create in the test tube can be used as evidence to support the "RNA World" theory for the origin and early evolution of life. For example, the fact that so many riboswitches are present today proves that RNA can compete well with protein genetic factors to sense metabolites and control gene expression despite the unforgiving forces of evolution. Likewise, the fact that numerous large noncoding RNAs are being revealed by bioinformatics searches of genomic DNA suggest that far greater numbers of sophisticated ribozymes exist and are entrusted by some cells to perform key biochemical tasks. Although pure RNA World creatures may have been driven to extinction billions of years ago, traces of their molecular systems are still with us today where they perform essential roles in all domains of life. We are eager to discover other examples of RNAs that may have had their origins in the RNA World.

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Selected Publications

Cheah MT, Wachter A, Sudarsan N, Breaker RR (2007) Control of alternative RNA splicing and gene expression by eukaryotic riboswitches. Nature 447:497–501

Sudarsan N, Lee ER, Weinberg Z, Moy RH, Kim J N, Link KH, Breaker RR (2008) Riboswitches in eubacteria sense the second messenger cyclic di–GMP. Science 321:411-413.

Breaker RR. (2009) Riboswitches: from ancient gene-control systems to modern drug targets. Future Microbiol. 4:771-3.

Weinberg Z, Perreault J, Meyer MM, Breaker RR (2009) Exceptional structured noncoding RNAs revealed by bacterial metagenome analysis. Nature 462:656-9.

Lee ER, Baker JL, Weinberg Z, Sudarsan N, Breaker RR (2010) An allosteric self–splicing ribozyme triggered by a bacterial second messenger. Science (in press).



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