Bacteria; Biochemistry; Biology; Biotechnology; Fungi; Genetics, Microbial; Microbiology; Molecular Biology; Computational Biology; Genomics; Metabolomics
Nucleic acids carry out numerous tasks in organisms that range from the long-term storage and transfer of genetic information to molecular recognition and biological catalysis. It is now apparent that both RNA and DNA have a tremendous untapped potential for biochemical function that can be accessed using molecular engineering strategies. Existing ribozymes can be altered by using test tube evolution, and entirely new enzymes made of RNA or DNA can be isolated from pools of trillions of sequence variants. In addition, we are finding that some types of functional RNAs though to be extinct are present in modern cells where they perform fundamental biochemical tasks. For example, we are discovering new examples of “riboswitches” that sense metabolites and control gene expression. We will continue to explore the functional potential of RNAs and DNAs that have been isolated from natural sources and that have been created outside the confines of cells.
Extensive Research DescriptionThe Breaker laboratory uses a variety of approaches to explore the fundamental properties of nucleic acids. For example, the laboratory develops new techniques for in vitro selection to create new functional RNAs and DNAs. In vitro selection is patterned after natural Darwinian evolution, but where "survival-of-the-fittest" is played out at the molecular level in the absence of living cells. Up to 100 trillion different molecules can be subjected to this test-tube evolution process to isolate or engineer molecules that perform tasks such as catalysis and molecular sensing.
Previous molecular engineering projects have provided evidence that both RNA and DNA have substantial untapped potential for sophisticated biochemical function. For example, we have produced a variety of new DNA enzymes, some that operate under cell-like conditions and perform reactions that mimic important biochemical transformations. In addition, we have generated dozens of examples of RNAs that function as designer molecular switches that respond to specific small molecules. These findings demonstrate that the primary roles of RNA and DNA in nature might be greater than currently appreciated, and suggests that the function of nucleic acids could be expanded via molecular engineering.Inspired by these molecular engineering demonstrations, we have more recently begun to search for novel types of non-coding RNAs that perform undiscovered catalytic or molecular sensing tasks in cells. We have identified numerous classes of "riboswitches", which are metabolite-binding mRNA domains that control genes responsible for biosynthesis of essential compounds. Among the first dozen riboswitches classes identified are representatives that sense coenzymes, nucleobases, amino acids or sugars. Some riboswitch classes exhibit complex biochemical behaviors including ribozyme activity, cooperative ligand binding, and logic gate function. In addition, we have identified other non-coding RNAs that are not riboswitches, but whose biological functions remain to be established. We will continue to use bioinformatics, genetics, and biochemistry techniques to discover new types of non-coding RNAs and to establish the functions of these complex-folded nucleic acids.The Breaker laboratory is working to discover novel non-coding RNAs in all three domains of life. Bioinformatics systems are used to identify candidate structured RNAs, and the functions of these new-found RNAs are validated using genetic and biochemical techniques.
In addition, the Breaker laboratory is exploring the functional capability and utility of nucleic acids when engineered outside the confines of cells.
- A. Roth, Z. Weinberg, A. G. Chen, P. Kim, T. D. Ames, R. R. Breaker (2014) A Widespread Self-cleaving Ribozyme Class is Revealed by Bioinformatics. Nat. Chem. Biol. 10:56-60.
- J. W. Nelson, N. Sudarsan, K. Furukawa, Z. Weinberg, J. X. Wang, R. R. Breaker (2013) Riboswitches in Eubacteria Sense the Second Messenger c-di-AMP. Nat. Chem. Biol. 9:834-839.
- A. Roth, R. R. Breaker (2013) Integron attI Sites, Not Riboswitches, Associate with Antibiotic Resistance Genes. Cell 153:1417-1418.
- H. Gu, K. Furukawa, Z. Weinberg, D. F. Berenson, R. R. Breaker (2013) Small, Highly Active DNAs that Hydrolyze DNA. J. Am. Chem. Soc. 135:9121-2129.
- J. L. Baker, N. Sudarsan, Z. Weinberg, A. Roth, R. R. Breaker (2012) Widespread Genetic Switches and Toxicity Resistance Proteins for Fluoride. Science 335:233-235.
- J. G. Wallace, Z. Zhou, R. R. Breaker (2012) OLE RNA Protects Extremophilic Bacteria from Alcohol Toxicity. Nucleic Acids Res. 40:6898-6907.
- E. R. Lee, J. L. Baker, Z. Weinberg, N. Sudarsan, R. R. Breaker (2010) An Allosteric Self-splicing Ribozyme Triggered by a Bacterial Second Messenger. Science 329:845-848.